P < .001) in 62 newborns with confirmed sepsis (3.1 ± 1.0 μg/mL) than in either 29 noninfected subjects (1.1 ± 0.3 μg/ml) or 110 healthy controls (0.91 ± 0.58 μg/ml). The diagnostic accuracy of serum calprotectin was greater  than that of the traditional markers of sepsis. In conclusion, serum calprotectin is an accurate marker of sepsis in very low birth weight newborns.To determine the diagnostic utility of serum calprotectin, a mediator of innate immune response against infections, we performed a multicenter study involving newborns with a birth weight <1500 g and a postnatal age >72hours of life. The diagnostic accuracy of serum calprotectin was compared with that of the most commonly used markers of neonatal sepsis . We found that the serum calprotectin concentration was significantly higher ( The neonatal immune response to many pathogens is largely immature . At thisNeonatal septicaemia is associated with hyperinflammatory host responses that subtend activation of immune system. A broad spectrum of inflammatory markers has been proposed for the diagnosis of neonatal sepsis . HoweverCalprotectin , a major product of innate immunity cells, is an antimicrobial peptide that protects cells against invasive microorganisms and regulates adhesion of leukocytes to the endothelium and extracellular matrix during the inflammatory process . CalprotCalprotectin has been proposed for the diagnosis of many inflammatory conditions . HoweverThis prospective investigation involved 3 Neonatal Intensive Care Units (NICUs) in the south of Italy over a 24-month enrolment study period. The study protocol was approved by the Ethics Committee of each center. Written informed consent was obtained from the parents of all infants enrolled in the study. The research was not sponsored by any company. Newborns were eligible for the study when they fulfilled all of the following criteria: (1) gestational age > 24 weeks; (2) birth body weight (BW) < 1500 g; (3) postnatal age > 72 hours; (4) no antibiotic treatment for at least 2 weeks before enrolment; (5) parental consent. Infants with the following conditions were excluded: (1) Apgar score < 3 at 5 min; (2) critical clinical conditions, as indicated by a blood pH < 6.8, or by the presence of hypoxia with persistent bradycardia; (3) incomplete clinical data or deviation from the study protocol; (4) maternal history of immunologic, inflammatory, or infectious diseases requiring antibiotic therapy during pregnancy, including the labour/delivery process; (5) surgery; (6) intraventricular haemorrhage grades III-IV . Enrolle2 requirement, bradycardia, unstable body temperature, unexplained and persistent metabolic acidosis (base deficit > 10 mmol/L), and/or hyperglycaemia (>10 mmol/L). White blood cell count (WBC), immature-to-total-neutrophil (I/T) ratio, platelet count, serum C-reactive protein (CRP), cultures of blood, urine, and endotracheal aspirate (when patient was intubated) for bacteria and fungi, chest or abdominal radiograph if the patient presented signs suggestive of thoracic or intra-abdominal disease, cardiac and brain ultrasonography, electroencephalography (EEG), and screening tests for metabolic diseases were performed in all subjects with suspected sepsis. According to standardized criteria , 13, 14,According to previously standardized criteria , 15, 16 Blood samples for SC determination were collected within 60 min of the onset of symptoms. For comparison, blood specimens were collected during routine sampling to determine SC levels from newborns without symptoms of sepsis, that is, the control group. The results of SC measurements were concealed until statistical analysis was concluded.μL of each sample was added to the wells of a plate and incubated at room temperature for 45 min. The plate was then washed 3 times with diluted washing solution, and 100 μL of purified rabbit anticalprotectin antibodies conjugated with alkaline phosphatase were added and incubated for 45 min at room temperature. A second washing procedure was performed, 100 μL of enzyme substrate solution was added to each well, and optical density was read at 405 nm. Serum calprotectin concentration was calculated from the standards and expressed as μg/mL [All laboratory investigations were performed by researchers unaware of the clinical data. Complete blood counts were done by cell counter . Leukocyas μg/mL –11. The The Pearson chi-square test was performed in the case of categorical variables. Continuous variables were expressed as mean and 95% confidence (CI). The groups were compared for variables by the one-way ANOVA procedure with the Bonferroni test for the posthoc analysis. After checking for assumptions, linear regression analysis with a stepwise method was used to study the possible effect of the different variables  on SC concentration. Binary logistic regression analysis was performed using as dependent variable the presence of sepsis and as independent variables WBC, I/T ratio, platelet count, CRP, and SC. P value of ≤.05 was considered significant. The optimal cut-off SC value to distinguish septic from nonseptic patients among subjects investigated for suspected sepsis was determined using receiving operating characteristic (ROC) curve analysis. Statistical analysis was performed using the SPSS software package for Windows  and StatsDirect (release 2.6.6).All tests of significance were two sided. A 12 E. coli, 8 Enterococcus faecalis, 6 Pseudomonas aeruginosa, 6 Klebsiella pneumonia, 6 Staphylococcus coagulase negative, 6 Candida albicans, 5 Serratia marcescens, 3 Streptococcus pyogenes, and 10 high probable sepsis), 29 were included in nonseptic group, and two infants were lost to followup . One hundred and ten infants were enrolled as controls. The main clinical and demographic characteristics were similar in the three groups , intraventricular haemorrhage (8 patients), critical clinical condition (8 patients), and lack of parental consent (2 patients). Ninety-three newborns  were investigated for suspected sepsis: 62 were classified as “septic”  had SC levels above normal value, whereas only 2/6 had deranged values of traditional markers . The diagnostic accuracy of all tests evaluated is reported in P < .001) and CRP  were predictors of sepsis in our population. The optimal cut-off serum calprotectin value to distinguish infants with sepsis from infants without sepsis was 1.7 on power . At thisA high sensitivity and negative predictive value, associated with a good specificity and positive predictive value, supports the use of SC in the diagnosis of sepsis in a VLBW newborn. A limited number of pathogens are responsible for sepsis among neonates. Most pathogens are encapsulated bacteria . The immAcute phase proteins, such as CRP , and theIn this context, SC measurement could overcome many of the limitations of the most common markers proposed for the diagnosis of neonatal sepsis, that is, haematological tests, acute phase proteins, chemokines, cytokines, and adhesion molecules . HaematoWe found that SC is an early, accurate, and easy-to-use marker of neonatal sepsis. When measured upon the appearance of the first clinical signs of sepsis, its sensitivity, PPV, and NPV were higher than those reported for traditional tests –4. SerumThe encouraging results reported herein provide a rationale for investigations of the role of SC in the management of sepsis. In particular, it may be worthwhile to assess the prognostic role of SC as regards the severity of sepsis. The high NPV of SC suggests that it could be an aid in therapy decision-making. In this context, the optimal cut-off value defined in this study could be useful in further research in this field. Finally, SC could help to resolve the dilemma of whether to start antibiotics immediately after sepsis screening and when to stop treatment, but there is a need for studies specifically designed to address these issues. The study has some limitations. Firstly, as reported in other studies , 13–16, To conclude, ours is the first study to investigate an innate immunity component as a biomarker of septicaemia in VLBW infants. Serum calprotectin could be a practical marker in the diagnostic approach to VLBW infants with suspected sepsis, and it could open the way to investigations exploring the role of other components of innate immunity in the diagnostic workup of neonates with infectious conditions.The authors declare that there is no conflict of interests.


To assess the relationship between muscle mass, body composition and muscle quality traits, we also analysed intramuscular fat content (IMF), water holding capacity (WHC), and additional physiological parameters in M. quadriceps and M. longissimus in 308 G3-animals.This study is aimed at the analysis of genetic and physiological effects of myostatin on economically relevant meat quality traits in a genetic background of high muscularity. For this purpose, we generated GM. longissimus (−0.13%) and M. quadriceps (−0.07%). No effect was found for WHC. A large proportion of variation of intramuscular fat content of the M. longissimus at the myostatin locus could be explained by sex (23%) and direction-of-cross effects (26%). The effects were higher in males (+0.41%). An additional locus with negative over-dominance effects on total fat mass (−0.55 g) was identified on chromosome 16 at 94 Mb (86–94 Mb) which concurs with fat related QTL in syntenic regions on SSC13 in pigs and BTA1 in cattle.We found that individuals with larger muscles have significantly lower total body fat (r = −0.28) and IMF (r = −0.64), and in females, a lower WHC (r = −0.35). In males, higher muscle mass was also significantly correlated with higher glycogen contents (r = 0.2) and lower carcass pH-values 24 hours after dissection (r = −0.19). Linkage analyses confirmed the influence of the myostatin mutation on higher lean mass (1.35 g), reduced body fat content (−1.15%), and lower IMF in The data shows QTL effects on mouse muscle that are similar to those previously observed in livestock, supporting the mouse model. New information from the mouse model helps to describe variation in meat quantity and quality, and thus contribute to research in livestock. Mstn, or growth and differentiation factor 8 - Gdf8) was first identified in knock-out mice as being a gene responsible for regulation of muscle growth [In livestock production, there is a high interest in controlling meat quantity and quality; knowledge about genes affecting muscle size and other meat properties can help breeders to select animals according to desired traits. Myostatin , which inhibits myostatin, are currently under review as therapeutics [Due to its large implication, not only on skeletal muscles, a lot of research has been undertaken on the effects of myostatin and its possible applications in health care and livestock production. The diseases ,14, carddiseases ,16. In ldiseases . In accoapeutics ,19.3-populations of reciprocal crosses between two hypermuscular Berlin Muscle Mouse Inbred (BMMI) lines to examine the genetic characteristics of myostatin and to find additional genes that could influence muscle growth and composition. The BMMI lines were hypermuscular as a result of long term selection that mirrors the selection process for high meat yield in livestock. The parental BMMI866 line originates from a population with the “compact” phenotype and therefore carries the known MstnCmpt-dl1Abc mutation [MstnCmpt-dl1Abc mutation on muscle mass have been identified on chromosomes 3, 5, 7, 11, 16, and X in a cross between Comp9 and CAST/Ei lines [In the present study, we generated Gmutation ,21. Althmutation . PreviouEi lines .Given that the BMMI lines were originally developed as supporting genetic models for livestock research, we were particularly interested in myostatin effects on intramuscular fat content (IMF) and water holding capacity (WHC) on the genetic background of high muscularity. We also analysed the extent to which sex and the direction of the reciprocal cross impacted on the traits of interest. The latter could indicate parent-of-origin effects, where the impact on the phenotype can be different depending on the parent from which an allele was inherited. For example, the polar over-dominance caused by the ovine callipyge locus, where a hypermuscular phenotype only occurs if the mutated allele is inherited from the sire ,25. Pare3-population. The linkage study did not reveal genomic loci accounting for variation of those metabolic traits.In addition to the relationship between muscle mass and meat quality traits, we were also interested in certain parameters of the muscle and whole body metabolism such as muscle glycogen and lactate contents, blood glucose levels, and the carcass pH-values. For this purpose, we present the correlations between these traits in the GMstnCmpt-dl1Abc mutant BMMI866 animals showed 42%, 42%, 99% and 94% higher values for body weight, lean mass, M. longissimus and M. quadriceps masses than the BMMI806, respectively. BMMI866 mice had 30% lower total fat percentage than BMMI806. The IMF contents of the M. longissimus and the M. quadriceps were 52% and 40% lower, respectively, as compared to the BMMI806 line. These data confirm the hypertrophic effect of the MstnCmpt-dl1Abc mutation and its impact on fat accumulation [post-mortem compared to BMMI806 (p < 0.05).As shown in Table mulation . Further1 and G3-population with the parental lines, we found intermediate values for body weight, lean mass and muscle masses. Dominance of the BMMI806 characteristics was indicated for total fat percentage in the F1 population. While the IMF values of both sexes of F1 animals and the males of the G3 population were intermediate, in female G3 animals, the measurements of total fat percentage and IMF of the M. longissimus were similar to the lower values of the BMMI866 line. Fasting blood glucose levels indicated a dominance of the BMMI866 line alleles, whereas the BMMI806 phenotype was found prevalent for the higher carcass pH-values for 1 hour post mortem.Comparing the F3-population, high positive correlations in males and females were observed between muscle mass and total lean mass (r > 0.79) and negative correlations with body fat percentage (r < −0.46) and IMF , whilst in males, no correlation was found. Furthermore, in males, higher muscle mass was associated with lower pH-values 24 hours post mortem (r < −0.16). Nevertheless, in both sexes negative correlations were evident between muscle glycogen contents and the pH-values of 1 and 24 hours post mortem (r < −0.15), and between WHC and the pH-value after 24 hours (r < −0.14). The muscle glycogen content was associated with the body fat content (r < −0.17), but was not correlated with the blood glucose levels; higher muscle glycogen contents were found in leaner animals.In the GM. longissimus in males supports the model of a metabolic shift towards glycolysis. The non-significant correlation with fasting blood glucose levels is likely due to the short fasting period of two hours before measurements leading to a high variability.The results regarding the carcass pH-value and WHC confirm studies in pigs, where a low pH-value negatively affects WHC and leads to high drip loss. The low WHC is likely the result of a developing acidic environment that affects the proteolysis of cell scaffold proteins ,32. In t3-population, we could only detect regions with larger effects. Two genome-wide significant QTL were identified  as causative for variation in total fat mass in mice.An additional QTL was identified on Chr16 at 94 Mb  13 (129–145 Mb). According to the animal genome database  this region contains QTL for marbling, back fat and for IMF [The QTL for fat mass on Chr16 is located within a region from 75–98 Mb that is syntenic with a region in pigs on  for IMF -35. Domi for IMF -38. Thus for IMF -42. A po for IMF . Finally for IMF ,44. AssuAs we did not find any other loci affecting muscle mass apart from the myostatin locus, we assume that several or many small effect alleles that contribute to these traits and that have been shown in other crosses  are hiddM. longissimus and M. quadriceps by 8.43 g, 5.74 g, 0.10 g and 0.07 g, respectively. In males, the total fat mass and the total fat percentage were also raised by 1.98 g and 2.97 points, respectively. Sex also affected IMF values of the two examined muscles, which were increased by 0.28 points and 0.41 points for the M. longissimus and the M. quadriceps in males, respectively. The increase in the lean mass related traits was expected as a sex difference; however, the increase in fat mass percentage in male mice was a new observation. An explanation could be that male BMMI806 mice display very high fat values . Animals derived from mating a female BMMI806 with a male BMMI866 showed higher body weight, lean mass and muscle mass at the Chr1 locus in comparison to animals of the reciprocal cross. While total fat mass, fat percentage and IMF of the M. longissimus were not significantly affected, we observed a large increase of IMF in the M. quadriceps by 0.29 points in male G3-animals that were derived from a mating between a female BMMI866 and a male BMMI806  and 21 (BMMI806) of inbreeding.The Berlin Muscle Mouse population had been long term selected for high body weight and high muscle mass to reflect the selective mechanisms in livestock breeding. Founder animals of the Berlin Muscle Mouse (BMM) population were originally purchased in several pet shops in Berlin, Germany. The selection process comprised several distinct phases. The beginning of the selection process constituted a phase of 23 generations of selection for high protein content of the carcass at the age of 60 days. Protein content was determined by chemical analyses. In a second phase, mice were selected for high body weight and low fat content at 42 days for 10 generations. Afterwards, mice were monitored for high muscularity by palpating and mice with highest muscularity on a scale between 1 and 5 were selected for the next generation. As a result of selection and likely the occurrence of natural mutation(s) over 25 generations, a mouse population with a high muscular phenotype had been generated. A high compact sub-line was perpetuated through random mating of selected animals . Sequenc3-intercross populations. F2-animals within every direction-of-cross were randomly mated under avoidance of sibling-mating [3-animals. Altogether, there were 195 males and 113 females. From these 308 animals, 97 males and 53 females came from the cross between a female BMMI866 and a male BMMI806 mouse, while the reciprocal cross between a male BMMI866 and a female BMMI806 mouse consisted of 98 males and 60 females. Using this design, we would expect different mitochondria and Y-chromosomes segregation in the reciprocal crosses. Since high-density SNP data of the inbred strains BMMI866 and BMMI806 provided evidence for no variation in both mitochondria and Y-chromosomes, we assume that the two parental mouse strains have the same mitochondria and Y-chromosomes as a result of their breeding history .Two pairs of full sibs of the Berlin Muscle Mouse inbred lines BMMI866 and BMMI806 were crossed reciprocally to generate F1, F2 and Gg-mating , to genead libitum access to food and water. Until the age of 70 days, the animals were fed a standard breeding diet . This diet contained 27.0% crude protein, 5.0% crude fat, 4.5% crude fibre, 6.5% crude ash, 50.5% nitrogen free extract (starch and sugar), vitamins, trace elements and minerals .The mice were treated in accordance to, and all experimental protocols were approved by, the German Animal Welfare Authorities . The animals were maintained under conventional conditions at 22 ± 2°C and controlled lightning with a 12:12 hour light:dark cycle. They were kept in groups of two to four animals of the same sex per Macrolon cage and given Musculus longissimus (ML) and Musculus quadriceps (MQ) were dissected and weighed. The summed muscle weights of the left and right M. longissimus and the left and right M. quadriceps were recorded as muscle mass (MM). The right muscles were immediately frozen in liquid nitrogen and subsequently stored at −80°C. The left muscles were cooled down to 6°C for one hour, subsequently frozen at −20°C and stored at the same temperature until WHC and IMF were measured. Carcasses were stored at 6°C and pH-values were taken within the M. biceps femoris at one and 24 hours post mortem . For the determination of WHC, frozen muscles were thawed and stored at 6°C for 24 hours. Muscles were then centrifuged for 60 sec at 604 × g in invitek® 1.5 ml receiver tubes with filter inlays to collect the tissue fluid that was not held from the muscle . The ratio of lost tissue fluid to tissue mass was designated as ‘drip loss’. IMF was measured as percentage of muscle weight using nuclear magnetic resonance technology  [M. longissimus  as suggested by Barham and Trinder (1972). Lactate contents were determined colorimetrically in the right M. longissimus using the Lactate Assay Kit by Techung Lee [After a fasting period of two hours, 71 day-old mice were anaesthetised under isoflurane and decapitated. The Germany) . Body weGermany) ,52. Bodyhung Lee .2 and the G3-animals  as implemented in GenABLE [p < 0.05) and LOD = 3.19 (p < 0.10), respectively. Genome-wide significant QTL (p < 0.05) were included into a mixed model for each trait to calculate the respective trait variance in the G3-population. Factors being significant under p < 0.001 were kept in the model. The genotype of the myostatin locus was accounted for by including it as a covariate in scans for additional QTL. Basic statistics were performed using the SAS software package . QTL support intervals were determined by bootstrapping as implemented in R/qtl [For the QTL analysis, 308 Gng R/qtl . Unlike  GenABLE -60. The  GenABLE . On averThe authors declare that they have no competing interests.SK conceived of the study, carried out the data collection, molecular genetic studies, statistical analysis of the data and drafted the manuscript. EMS and AOS participated in the statistical analysis, particularly in correcting the data for population stratification and estimating heterozygosity, respectively. AW carried out lactate and glucose measurements, FPMV and HY provided the data of the mouse diversity array (MDA). GAB participated in conceiving of the study, its design and coordination and helped drafting the manuscript. All authors read and approved the final manuscript.


Anaemia is a known risk factor for cardiovascular disease and treating anaemia in chronic kidney disease (CKD) may improve outcomes. However, little is known about the scope to improve primary care management of anaemia in CKD.An observational study  with a nationally representative sample using anonymised routine primary care data from 127 Quality Improvement in CKD trial practices (ISRCTN5631023731). We explored variables associated with anaemia in CKD: eGFR, haemoglobin (Hb), mean corpuscular volume (MCV), iron status, cardiovascular comorbidities, and use of therapy which associated with gastrointestinal bleeding, oral iron and deprivation score. We developed a linear regression model to identify variables amenable to improved primary care management.The prevalence of Stage 3–5 CKD was 6.76%. Hb was lower in CKD (13.2 g/dl) than without (13.7 g/dl). 22.2% of people with CKD had World Health Organization defined anaemia; 8.6% had Hb ≤ 11 g/dl; 3% Hb ≤ 10 g/dl; and 1% Hb ≤ 9 g/dl. Normocytic anaemia was present in 80.5% with Hb ≤ 11; 72.7% with Hb ≤ 10 g/dl; and 67.6% with Hb ≤ 9 g/dl; microcytic anaemia in 13.4% with Hb ≤ 11 g/dl; 20.8% with Hb ≤ 10 g/dl; and 24.9% where Hb ≤ 9 g/dl. 82.7% of people with microcytic and 58.8% with normocytic anaemia (Hb ≤ 11 g/dl) had a low ferritin (<100ug/mL). Hypertension (67.2% vs. 54%) and diabetes (30.7% vs. 15.4%) were more prevalent in CKD and anaemia; 61% had been prescribed aspirin; 73% non-steroidal anti-inflammatory drugs (NSAIDs); 14.1% warfarin 12.4% clopidogrel; and 53.1% aspirin and NSAID. 56.3% of people with CKD and anaemia had been prescribed oral iron. The main limitations of the study are that routine data are inevitably incomplete and definitions of anaemia have not been standardised.Medication review is needed in people with CKD and anaemia prior to considering erythropoietin or parenteral iron. Iron stores may be depleted in over >60% of people with normocytic anaemia. Prescribing oral iron has not corrected anaemia. Anaemia is an independent risk marker for cardiovascular morbidity and mortality and it is a common complication in patients with chronic kidney disease (CKD) -3. CKD iPatients with CKD have a high cardiovascular risk and effective anaemia management may improve outcomes -17. PartDespite advances in the use of ESA for CKD patients, there is low uptake of this therapy . AlthougWe carried out this cross-sectional study to report the prevalence of anaemia in CKD, examine its association with cardiovascular diseases, and assess whether practitioners had attempted to treat the patients with oral iron.We used data from the Quality Improvement in Chronic Kidney Disease (QICKD - ISRCTN5631023731) trial ,33. The 2 as having a normal eGFR, those with eGFR ≥60 ml/min/1.73 m2 and <90 ml/min/1.73 m2 as having “mildly impaired renal function.” We subdivide those with an eGFR <60 ml/min/1.73 m2 into stages 3A, 3B, 4 and 5 using conventional eGFR ranges [We defined cases of stages 3 to 5 CKD by the estimated glomerular filtration rate (eGFR), taking into account the requirement of three month period of chronicity for formal diagnosis. Wherever available we used laboratory calculated eGFR because it is subject to a national calibration scheme . We descR ranges . Where wR ranges .We extracted a dataset that included Hb, mean corpuscular volume (MCV) and common potential causes of anaemia so that we could differentiate between renal and other causes of anaemia. We defined anaemia as Hb ≤ 11 g/dl in line with NICE guidance .We report the prevalence of Hb measurement among people with CKD and compare their Hb with those in people without CKD. We also explored what proportion of people had a recent measure of Hb, which we define as a recording within the last two years. We classify anaemia into micro-, normo- and macrocytic based on the MCV. Microcytic anaemia is defined as an MCV of <80 fl, normocytic as 100-80 fl, and macrocytic as >100 fl . We alsoTo explore the association between age and anaemia the prevalence of anaemia by eGFR category and age band we report the change using previously described groupings .We explored any association between CVD, CKD and anaemia. We included in our definition of cardiovascular disease (CVD) diagnoses of heart failure (HF), ischaemic heart disease (IHD), stroke, transient ischaemic attack and cerebrovascular disease (CEVD), peripheral vascular disease (PVD), and hypertension (HT); and diabetes mellitus.We investigated whether anaemic patients had been prescribed aspirin and non-steroidal anti-inflammatory drugs (NSAIDs), clopidogrel or warfarin. These are medications which are all associated with an increased risk of gastrointestinal bleeding.Finally we looked at oral iron prescriptions in order to see if iron was being prescribed to correct anaemia and to assess whether its use was associated with correction of anaemia. As intravenous iron and ESA therapy is largely provided via specialist renal units, information about parenteral iron is not contained within the family practice computerised medical record.The data used in the study were extracted from primary care computer systems and processed using our established method ,47. We aB); it standard error (E); and significance (p). B represents the unit change in the outcome variable for either one unit of change, or the presence or absence of the predictor variable. E.g. eGFR has a B of 0.003, this means that for every 10 ml/min rise in eGFR Hb rises by 0.03 g/dl; B Stage 3 to 5 CKD is −0.482, implying that people with stage 3 to 5 CKD have an Hb 0.5 g/dl lower than those who do not. We did not include age, gender or ethnicity in our model as they are already included in the equation used to estimate renal function [We carried out a regression analysis initially testing each variable separately to explore any predictive effect on our outcome variable (Hb). We then grouped our best predictor variables into a single model reporting the unstandardised coefficient  .The prevalence of CKD in the general population is 5.3% n = 58,592); 6.76%  in people aged over 18 years. As renal function declines from normal to stage 3B CKD the mean age of the people gets older with each declining stage of CKD; those with stage 4 and 5 CKD are slightly younger , compared to 13.22 g/dl  in patients with CKD. Furthermore, prevalence of anaemia was increased in the higher CKD stages  of people with CKD have had their ferritin measured. In two-thirds  of these patients the ferritin is <100ug/mL and 2.7%  had a ferritin level <15ug/mL; suggesting that they had reduced iron stores. Over three-quarters (82.7%) of people with CKD and microcytic anaemia (Hb ≤ 11 g/dl) have a ferritin less than 100 ug/mL , compared with 58.8% of those with normocytic (Chi-square p < 0.001) and 45.5% of those with macrocytic anaemia . Furthermore, 39% patients with CKD and microcytic anaemia have ferritin level below 15 ug/mL , heart failure (HF), stroke and transient ischaemic attack grouped as cerebrovascular disease (CEVD), peripheral vascular disease (PVD) and diabetes is approximately twice that of CKD patients without anaemia Table . In addiTwo-thirds of patients (62%) in our study receive one or more drugs causing anaemia in the last two years, 84.7% ever. Nearly half of people with CKD and anaemia have been prescribed aspirin and non-steroidal anti-inflammatory drugs (NSAID) , 8.1% (n = 378) received clopidogrel and 10.3% n = 485) warfarin in the last two years. Analysis of medication history showed that 61%  of people with CKD and Hb ≤ 11 g/dl had taken aspirin, three-quarters  NSAID, 14.1% (n = 662) were taking warfarin and 12.4% (n = 581) clopidogrel. Furthermore, more than half  of patients with iron deficiency anaemia and CKD stage 3–5 were concurrently prescribed NSAIDs and aspirin  of people with anaemia in the general population had been prescribed oral iron therapy (Table 2.6% of pTwo-thirds (67.6%) of people with anaemia and CKD and a low ferritin level were taking oral iron. The mean Hb in the iron treated group was 10.0 g/dl (n = 582) compared with 10.3 g/dl (n = 1214) in the non-iron treated (t-test p < 0.001).2 = 0.9%). The regression analysis testing each variable separately showed a small but significant effect of eGFR and CKD on the outcome variable, haemoglobin . Our best model included: CKD stages 3–5, significant proteinuria, heart failure, diabetes, hypertension, stroke, deprivation score and NSAID within last 2 years, R2 = 7.4% (p < 0.001). After adding other elements to the model the CKD remained a significant contributor to the final effect (Table 2 = 7.7%). We excluded ACE-I from our final model. The unstandardised coefficient (B) was smaller than that for hypertension and the overall model performed less well with ACE-I and hypertension included.Stage 3–5 CKD was associated with a reduction in Hb, 0.712 g/dL  population ,5. HowevStage 3–5 CKD is an independent predictor variable of reduced haemoglobin. The current focus of UK National guidance on anaemia management in CKD needs to be reviewed. We recommend a shift from early referral to specialist centres for administration of parenteral iron or ESA to more effective medication management in primary care, with improved access to parenteral iron. Family physicians should carefully balance the risk benefit ratio of prescribing aspirin in cardiovascular disease and of NSAIDs in people with CKD. The concurrent use of acid suppressant therapy may help reduce the risk of gastrointestinal blood loss.Our findings are consistent with a systematic review and meta-analysis that showed little benefit from oral iron in people with CKD. Interestingly, the rise in Hb reported using parenteral iron (0.83 g/dL) is similar to the decline seen in people with CKD (0.72 g/dL) .In USA the Third National Health & Nutrition Examination Survey Public health (NHANES III) reported a high prevalence of anaemia, and a low ferritin in people with heart failure and CKD, which is also compatible with our results . LikewisOur approach is limited by the inevitable incompleteness of the routine data, though their strengths and weaknesses are well known . The assA further difficulty is that definitions of anaemia are not completely standardised. The European guidelines define anaemia as <11 g/dl , which iSerum ferritin <100 ng/mL is used in this paper as a surrogate for iron deficiency; this is an expert consensus, used in UK national guidance  though mProspective studies are needed to assess whether more effective anaemia management strategies might reduce the incidence of CVD in people with CKD. Tools and algorithms are needed to help family doctors asses the relative risk of stopping NSAIDs, aspirin and other therapy against the potential benefits of correcting anaemia.Anaemia is common among CKD patients in the UK and associated with cardiovascular diseases and prescription of drugs which may cause anaemia. Having stage 3–5 CKD is a predictor variable of a decline in haemoglobin. Primary care management should start with a careful medication review and assessment prior to referral for replacement of depleted iron stores with parenteral iron.ACE-I: Angiotensin converting enzyme inhibitors; ARB: Angiotensin II receptor blockers; CEVD: Cerebrovascular disease; CKD: Chronic kidney disease; CVD: Cardiovascular disease; eGFR: Estimate of glomerular filtration rate; ESA: Erthyropoiesis-stimulating agents; FDA: Food and Drug Administration; Hb: Haemoglobin; HT: Hypertension; HF: Heart failure; IHD: Ischaemic heart disease; IMD: Index of multiple deprivation; LVMI: Left ventricular mass index; MCV: Mean corpuscular volume; NSAID: Non-steroidal anti-inflammatory drugs; NICE: National Institute for Health and Clinical Excellence; NHANES III: Third National Health & Nutrition Examination Survey Public health; PVD: Peripheral vascular disease; SPSS: Statistical Package for Social Sciences Version 18; QICKD: Quality Improvement in Chronic Kidney Disease; WHO: World Health Organization.http://www.nhsemployers.org/SiteCollectionDocuments/Chronic_kidney_disease_FAQs%20-%20ja040711.pdf. IMacD: Professor Macdougall has received consultancy and lecture honoraria from several companies involved in the manufacture of ESAs and IV iron products, and is a current Work Group member of the KDIGO Anemia Guidelines Group. HG: None. CT: None. KH: Kevin Harris has received funding from several pharmaceutical companies, Edith Murphy Foundation 2007–2010: Quality Improvement in CKD due to diabetes and LNR CLAHRC for Prevention of Chronic Disease and its Associated Co-Morbidity theme. TD: None. DG: Honoraria and Speaker Fees from Takeda, Roche, Sandoz, Amgen.OD: None. SdeL: Involved in development of the pay-for-performance indicator for UK practice; lead author for Department of Health Frequently Asked Questions about chronic kidney disease. OD, SdeL, ICM and DG conceived and designed the study; OD wrote the first draft of the manuscript, which SDEL developed. OD and SDEL performed the analysis. OD, SdeL, ICM, HG, CT, KH, TD and DG. All authors contributed to editing the paper, and approved the final version of the paper.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2369/14/24/prepubFigure S1. Age sex profile of study population compared to census data.Click here for fileFigure S2. Patients by stage CKD, age and sex.Click here for file


Purpose. We hypothesize that intra-abdominal hypertension (IAH) is associated with the presence of anaerobic metabolism in the abdominal rectus muscle (RAM) tissue of critically ill patients. Methods. We included 10 adult, critically ill patients with intra-abdominal pressure (IAP) above 12 mmHg. Microdialysis catheters (CMA 60) were inserted into the RAM tissue. The samples were collected up to 72 hours after enrollment. Results. The patients' median (IQR) APACHE II at inclusion was 29 (21–37); 7 patients were in shock. IAP was 14.5 (12.5–17.8) mmHg at baseline and decreased significantly over time, concomitantly with arterial lactate and vasopressors requirements. The tissue lactate-to-pyruvate (L/P) ratio was 49 (36–54) at the beginning of the study and decreased significantly throughout the study. Additionally, the tissue lactate, lactate-to-glucose (L/G) ratio, and glutamate concentrations changed significantly during the study. The correlation analysis showed that lower levels of pyruvate and glycerol were associated with higher MAP and abdominal perfusion pressures (APP) and that higher levels of glutamate were correlated to elevated IAP. Conclusions. Moderate IAH leads to RAM tissue anaerobic metabolism suggestive for hypoperfusion in critically ill patients. Correlation analysis supports the concept of using APP as the primary endpoint of resuscitation in addition to MAP and IAP. Intra-abdominal hypertension (IAH), defined as a sustained intra-abdominal pressure (IAP) above 12 mmHg, directly affects splanchnic perfusion and is one of the triggers for multiple organ failure . In up tWe previously demonstrated that short-term increase of IAP to 12 mmHg results in increased lactate-pyruvate (L/P) ratio in the abdominal rectus muscle (RAM) of laparoscopic surgical patients, which is detected using microdialysis (MD) . This fiThe study was approved by the University of Tartu Ethics Review Committee on Human Research (Protocol Number 170/T-11 28.04.2008). The study was performed according to the Declaration of Helsinki.This study was a prospective, single-center, and observational study.acute pancreatitis;liver failure with cirrhosis and ascites;gastrointestinal hemorrhage;use of vasopressors and inotropic agents;2/FiO2 ≤ 300 mmHg.PaOAdult, mechanically ventilated patients who were admitted to the Department of General Intensive Care (ICU) at Tartu University Hospital were screened for study inclusion during the first 3 days of their treatment. The patients were eligible for the study if they were ≥18 years of age, if the IAP measurement was possible , and if they had at least one of the following risk factors for the development and presence of IAH at the time of ICU admission:These criteria were previously identified as risk factors for IAH in adult, mechanically ventilated, ICU patients . The curn = 2 patients), gastrointestinal bleeding (n = 2 patients), ruptured abdominal aortic aneurysm, acute pancreatitis, multiple trauma, methanol intoxication, cardiac failure, and tetanus.After the patients provided informed consent, 10 patients  with a median age of 65  years were studied. The reasons for ICU admission were postresuscitative state . The system is based on a revised closed-system, repeated measurement technique . An inst2.Exclusion criteria were as follows: patient's or next-of-kin's refusal to participate, abdominal surgery, and BMI ≥ 32 kg/mμL/min. The sample specimens were collected at 26 time points: hourly for the first eight hours, every two hours for the next eight hours, and then every four hours until the end of the study. The duration of the study was 72 hours. The sample specimens were stored in a freezer at −80°C at Tartu University Hospital in Estonia and were sent in a single shipment to Tampere University Hospital in Finland for additional analyses. Glucose, lactate, pyruvate, glycerol, and glutamate concentrations of the microdialysates were measured using a CMA 600 analyzer . The lactate-to-pyruvate ratio (L/P ratio) and lactate-to-glucose ratio (L/G ratio) were calculated.At the beginning of a study, an MD catheter  was inserted into the RAM. Catheter placement was confirmed using ultrasound . The microdialysate perfusion rate was set at 0.3 Statistical analyses were performed using GraphPad Prism 5.02 . Normality of distribution was tested using the Kolmogorov-Smirnov test. For the RAM tissue metabolite concentrations , Friedman's test was used to test the change over the observation period. The patients' clinical characteristics were averaged for corporate 6 time points  and analyzed using a repeated-measurements analysis of variance  or Friedman's test. For the post hoc analysis, Dunn's test was used.P ≤ 0.05.For correlation analysis, consecutive single measurements were used. Analysis was performed using the STATISTICA 10  software; correlations within the subjects (using the method by Bland and Altman) were employed because of the presence of multiple measurements from one patient . To remoSeven of 10 patients were in shock at the beginning of the study: 2 patients were in septic shock, 2 patients were in cardiogenic shock, 2 patients were in hemorrhagic shock, and 1 patient was in distributive (methanol poisoning) shock. One patient died 16 hours after being included in the study; the remaining patients were discharged alive from the ICU. The median length of stay in the ICU was 11 (8–18) days. The patients' median APACHE II score at the time of inclusion in the study was 29 (21–37).The patients' clinical characteristics are presented in P = 0.002) (IAP was moderately elevated at baseline (14.5 12.5–17.8) mmHg) and decreased significantly over the observation period (= 0.002) . The mea= 0.002) , whereas= 0.002) ; however.5–17.8 mμM, and lactate was 4.7 (2.7–10) mM. The baseline L/P ratio was 49 (36–54) and the L/G ratio was 0.93 (0.67–1.7). The dynamics of RAM tissue metabolite concentrations during the study period are shown in The baseline value of tissue glucose was 4.1 (3.2–6.5) mM, pyruvate was 129 (57–189) P ≤ 0.008). Blood lactate was also elevated at the beginning of the study and normalized earlier than tissue lactate (P ≤ 0.02). The L/P ratio decreased significantly (P ≤ 0.0002) throughout the observation period and normalized 24 hours after enrollment in the study. The L/G ratio reached its maximum level at the 10th hour and subsequently started to decrease. Changes that occurred during the initial 36 hours were significant (P ≤ 0.006); however, the changes were not significant during the entire period of 72 hours.The blood and RAM tissue glucose were stable throughout the observation period . Tissue Correlation analyses revealed an association between higher MAP and APP levels and lower tissue pyruvate concentrations and between the noradrenaline dose and RAM tissue pyruvate concentrations . The eleμM and glycerol 251 (173–408) μM. The baseline concentrations of tissue glutamate and glycerol were as follows: glutamate 38 (22–100) P ≤ 0.0001). A correlation analysis indicated an association between elevated IAP levels and higher tissue glutamate concentrations ; however, the L/G ratio trend did not continue over the entire 72 hours of observation time (P ≤ 0.08).Glucose is the basic energy substrate for the cells. Setälä and Gudaviciene found that tissue glucose and lactate concentrations, especially their relation L/G ratio, may indicate the presence of ischemia in a microvascular flaps. A high L/G ratio was associated with the amount of flap necrosis . Our resGlutamate is a proteinogenic amino acid that may act as a neurotransmitter or metabolic substrate. Elevated extracellular concentrations may reflect excitotoxicity and membrane breakdown . GlutamaGlycerol, which is most likely released from damaged cells because of ischemia and tissue injury, is related to the degradation of the glycerophospholipids of cell membranes . In the At the beginning of study, 7 of 10 patients were in shock. From our microdialysis results, it is unclear whether the observed changes occurred because of shock resolution or because of dynamics in IAP and APP. Control samples of nonabdominal origin, for example, from the extremities, would have been desirable to address this question; however, due to ethical and cost concerns, we inserted only one MD catheter per patient. This factor is the primary limitation of our study. Indirect support to our findings is derived from animal experiments by Meier and Benninger, who demonstrated that other tissues/locations are not influenced by IAP as fast as RAM is. Furthermore, the positive correlation between glutamate and IAP indicates the importance of IAP in observed metabolite changes.The present study demonstrates that moderate IAH leads to RAM tissue anaerobic metabolism suggestive for hypoperfusion in critically ill patients. The correlation analysis supports the concept of using APP as a primary endpoint of resuscitation in addition to MAP and IAP. Feasibility of RAM microdialysis as a diagnostic tool for early-stage tissue damage requires additional investigation.


BMI and PBF varied significantly by race and gender . Asian-American women had the lowest BMI (21.5 kg/m2) but the second highest PBF (27.8%). Linear association between BMI and PBF was the weakest (r2 = 0.09) among Asian-American women and BMI had the poorest sensitivity (37%) to detect PBF. The high PBF with low BMI pattern exhibited by Asian-American women suggests that they could escape detection for obesity-related disease if BMI is the sole measure that estimates body composition.Obesity prevalence and average body composition vary by US race and gender. Asian Americans have the lowest prevalence of obesity. Relying on body-mass index (BMI) to estimate obesity prevalence may misclassify subgroups that appear normally weighted but have excess body fat. We evaluated percentage body fat (PBF) and BMI to determine whether BMI reflects PBF consistently across different races. 940 college students were recruited from a local public university over four consecutive years. We measured PBF by bioelectrical impedance analysis (BIA), weight by physicians' scales, and height with stadiometers. Our sample comprised Asians (49%), Caucasians (23%), Hispanics (7%), and Other (21%). Participants averaged 21.4 years old; BMI was 22.9 kg/m Body-mass index (BMI), an important indicator of obesity prevalence in large populations, generally reflects degree of fatness among individuals. Body-mass index can however over- or underestimate adiposity depending upon certain circumstances.Accurate determination of obesity has become exceedingly important because of major health threats posed by excess adiposity. Obesity is associated with increased incidence of cardiovascular disease, diabetes, sleep apnea, degenerative joint disease, and site-specific cancers –6. MoreoValidation studies have evaluated accuracy of BMI in estimating body fatness, by comparison to more refined measures such as bioelectric impedance analysis (BIA) and dual energy absorptiometry (DXA) –21. SomeThe prevalence of inactivity has increased among all age groups and is thought to be a major contributor to the obesity epidemic . With thSerial cross-sectional samples of college-age men and women were recruited during winter quarter of four consecutive years, 2006, 2007, 2008, and 2009 from an undergraduate physiology course at a major public university in Southern California. All 1029 students in the course over the three years were eligible and subsequently enrolled  into the study. Data from 940 students with complete covariate information were included.The study was approved by the Institutional Review Board of the University of California at Los Angeles. We certify that all applicable institutional and governmental regulations concerning the ethical use of human volunteers were followed during this research.Ethnicity and racial background were self-identified using the following categories: White/Caucasian; Hispanic; Black/African American; Middle Eastern; Indian; Native American; Asian (Eastern); Asian (Southeast); Pacific Islander; and Other. A blank space to write in “Other” was provided, and, additionally, multiple categories could be marked in the event of mixed race or ethnicity. Age was self-reported.Because of small numbers of individuals in some of the racial/ethnic groups and to increase the power of our stratified analyses we combined race/ethnicity into four groups: Asian, White, Hispanic, and Other. Eastern and Southeastern Asian groups were combined into Asian. Other category consisted of multiple ethnicities, recorded Other, Middle Eastern, Native American, African American, and Pacific Islanders. Self-reported White/Caucasian and Hispanic were retained as singular categories.Trained clinical technicians conducted all anthropometric measurements. Subjects were weighed while wearing no shoes. Body weight was measured with a physicians' scale. Heights were taken with a stadiometer .2) + (b × Wt) + (c × A) + (d × R) + e, where FFM is fat-free mass, Ht is height (cm), Wt is weight (kg), A is age (years), and R is impedance (Ω). The constants, a through e, are proprietary information of Biodynamics, Inc. We utilized the model for estimation of FFM for our entire study population. We constructed categories of body-mass index using the WHO International Criteria for all populations  [2; 24–27.5 kg/m2; 27.6–32.49 kg/m2; ≥32.5 kg/m2) [Bioelectrical impedance analysis (BIA), used to estimate percent body fat (PBF) and fat and fat free mass, was conducted with a quadripolar BIA device . Fat-free mass and fat mass are estimated with a regression equation based on data obtained through comparison with bioimpedance estimates with hydrodensitometry . The equation used by the present study, and also utilized in our earlier study , estimat0 kg/m2) , and the5 kg/m2) .Analysis of variance was used to evaluate differences in anthropometric variable means according to race and gender. We further analyzed associations between BMI and PBF by constructing multiple linear regression models adjusting for gender and ethnicity and linear regression models of BMI and PBF according to whether Asian or Caucasian separately for males and females. We plotted scatter distributions of BMI and PBF for Asian and Caucasian males and females.2) and PBF (≥25.0% for men and ≥30.0% for women) divided by the total classified as obese by PBF. We computed specificity as the proportion of participants classified as nonobese  by BMI (<30.0 kg/m2) and nonobese  by PBF (<25.0% for men and <30% for women) divided by the total classified as nonobese by PBF [To provide estimates of sensitivity and specificity of BMI to predict PBF, we evaluated distributions of gender and ethnic subgroups according to BMI and PBF. If we consider PBF measured by BIA as the gold standard, we computed sensitivity as the proportion of participants classified as obese by BMI . All reported 2, weight 65.4 kg (145 pounds), and height 169 centimeters (cm) (66 inches). Total PBF estimated by BIA was 25%. Fat mass averaged 17 kilograms (kg); fat-free mass 49 kg.Most study participants see  were Asi2 and 25 kg/m2, respectively, while Hispanic females (mean = 30%), Asian females (mean = 28%), and Other females (mean = 29%) had the highest percentage body fat. Asian-American males (mean = 174 cm) (68 inches) and Asian-American females (mean = 161 cm) (63 inches) were the shortest among the racial/ethnic subgroups.Almost all anthropometric measures were significantly different according to race and according to gender and race see . Hispanir2 = 0.52) in PBF was explained by BMI, ethnicity, and sex. We further compared degree of association between BMI and PBF for both Asian-Americans and Whites by constructing separate scatter plots for males and females  and less precisely associated for Whites (r2 = 0.34). Among females however the association was weaker, particularly among Asian-Americans (r2 = 0.09).Results from multiple linear regression analyses suggested that 52% of the variability (2) and the WHO Asian (BMI ≥ 27.5) cutpoints for obesity [We utilized the WHO International  and women (26.5 kg/m2) between the ages of 20 are 29 is the lowest of all adult age groups younger than age 80 years [2 and females 22.0 kg/m2.We studied college-age young adults because this age group, in particular, may be more likely to have BMI measurements in the normal to intermediate range. The average body-mass index for US men (27 kg/m80 years . The ave2) and women (21.5 kg/m2) had the lowest mean BMI among the study sample, while Hispanic men (25.9 kg/m2) and women (23.5 kg/m2) had the highest mean BMI. Percentage body fat did not follow the same distribution pattern however. While Asian women had the lowest BMI, they did not have the lowest percentage body fat. Asian women had 27.8% body fat, while Caucasian women, lower than Asian women, had 26.9%. Hispanic women had the highest percentage body fat (29.8%).Estimates for body composition among the college-aged sample were strikingly different for gender and race. Asian men . Our correlation for men was similar to a study using the Third NHANES sample . Their sAge, gender, and ethnicity have been found in several studies to affect strength of relationship between BMI and percentage body fat , 17–21. Gender also affects the degree to which BMI predicts body fat , 25, 29.r2 = 0.47) than for Caucasian men (r2 = 0.34), while the reverse was true for women. The association between BMI and PBF was the weakest for Asian women (r2 = 0.09) compared to Caucasian women (r2 = 0.36).The relationship between PBF and BMI has been shown to differ according to ethnic origin . A meta-High percentage of body fat occurring at lower BMIs has also been observed among younger Japanese in a multinational study of Japanese, Caucasians, and African Americans conducted in Japan, the United Kingdom, and the United States . The stu2) that we observed [The low degree of association between BMI and PBF that we observed for young Asian-American women in particular may signal a present and future risk for obesity-related disease. BMI was a poor predictor of PBF in Asian-American women reflected by a low sensitivity (37%). The low sensitivity and weak association suggest that use of BMI to estimate adiposity may be especially inaccurate in Asian-American women. In a comparative study of body composition in Asian and Caucasian young adult females, results showed a similar PBF (31%) for Taiwanese women aged 20 to 29, with a similar BMI (23.7 kg/mobserved . In a coobserved . In a laobserved .Our reliance on BIA to estimate PBF measurement may have contributed to potential inaccuracies in our data. A validation study of body fat estimation by BIA compared to DXA conducted among multiethnic women showed that underestimation of lean body-mass was affected by whether being Caucasian or African American , althougIn conclusion we observed striking differences in body composition according to gender and ethnicity among a young adult college-age population. While most males and females of different ethnicities had similar associations between PBF and BMI, Asian-American females represented a special subgroup where BMI did not accurately reflect underlying adiposity. The weight and BMI measurements were representative of normal; however the relative high PBF may put Asian-American females at risk for future obesity-related disease.


Bone marrow (BM) aspiration is largely used for relapse assessment in acute myeloid leukemia (AML). It remains unclear what roles that BM trephine biopsy plays on relapse assessment.Bone marrow (BM) sections during complete remission (CR) from 60 acute myeloid leukemia (AML) patients were retrospectively analyzed. Computer image processing technology was performed for detection of the distance between precursors and endosteum, and density of precursors was also calculated under light microscopic image. Immunohistochemistry was used to identify the immunophenotype of clustered precursors.2 for cluster density, early relapse was detected with a sensitivity of 51.5% and a specificity of 85.7%.Except for single and double precursors, there existed clustered precursors of 3-5 cells during CR. Here, we demonstrated that clustered precursors, but not single and double precursors, were useful in risk factor of relapse. Area under the receiving operator curve (ROC) was of 0.007 . Using a standard cut-off value of >4.0/mmMultivariate Cox regression analysis revealed that clustered precursor is an independent risk factor for early relapse .2 in sections. Early aggressive interventions are needed to prevent hematologic relapse.Cumulatively, clustered precursors in BM sections during CR may serve as an independent risk factor of early relapse and poor outcome for AML patients in cluster density > 4.0/mm In leukemia, the leading cause of treatment failure is disease relapse. Salvage treatments for relapsed patients frequently lead to dismal outcomes including low levels of complete remission (CR) and short overall survival (OS) times -4. HitheSimilar to the observations made for cell numbers, cell localization might also provide important information applicable to disease diagnosis and perhaps prognosis assessment. In normal human subjects, precursors are rare and are found localized near the endosteum, and consist of 1-2 cells. In some cases of myelodysplasia syndrome (MDS), immature precursors might be located in the intertrabecular region and occasionally aggregate as clusters which are of 3 ~ 5 cells, such clusters were initially defined as abnormal localization of immature precursors (ALIP) by Tricot . ALIP prIn the present study, we found during CR of AML patients, there existed clustered precursors consisting of 3 to 5 cells in the intertrabecular region. Since they were morphologically and anatomically analogous to ALIP in MDS, we defined them as ALIP-like clusters. The precise information that histological data provides during CR is largely unknown. Here, we extended our investigations to explore a histological relapse indicator by comparing the frequency of ALIP-like clusters between relapse and no-relapse cases.2, 7-8 mg/m2, (60–90) or (8–10) mg/m2, respectively) for 3 days and cytarabine at (100–150) mg/m2 daily for 7 days. For consolidation therapies, AML patients received up to 6 cycles of induction therapies including one alternative anthracycline with similar dosage mentioned above. All trephine biopsies and administrations were performed after having obtained informed consent. This study was approved by the institutional Ethics Committees of our hospital and conducted in accordance with the ethical guidelines of the Declaration of Helsinki. First check-up post induction was assessed by bone marrow biopsy on day 21–28 post induction. Concerning the physical condition of the patients, the biopsies might be retarded until peripheral blood recovery. During CR, BM biopsies were routinely performed before every consolidation therapies. After consolidation therapies, bone marrow was surveilled by biopsies every 3 months in the first year and every 6 months in the second year. BM sections and smears from the patients were assayed under paired analysis from the time of diagnosis to the follow-up. Cases in CR were divided into relapse and no-relapse groups according to patients relapsed or not during the follow-up. The pathologists who examined the BM samples were not participant of this study and were innocent about the group situation. Clinical outcome was assessed by relapse-free survival (RFS) and overall survival (OS).Between December 2004 and February 2013, 115 patients with de novo AML were admitted to the Hematology Department of Shanghai Sixth People’s Hospital, China. Patients were diagnosed according to the FAB classification. CR and relapse were diagnosed according to Cheson et al. [2.BM biopsy samples were collected from AML patients during CR and fixed in Bouin fixative. Dehydration was performed by exposing specimens to ascending concentrations of ethanol. All samples were embedded in Hemapun865 plastic. The component of the plastic and the detailed embedding technique had been previously described . BM sectTo study the cellularity of ALIP-like clusters, IHC staining was performed. The formalin-fixed, EDTA (PH 7.4) decalcificated and paraffin-embedded bone marrow tissues were cut at a section thickness of 3 μm. The staining procedure was done in accord with the instructions provided with an UltraVision Quanto Detection System HRP DAB kit’s . In brief, sections were deparaffinized in xylene and rehydrated by exposure of the specimens to graded ethanol. Antigen retrieval was performed by sodium citrate buffer with pH 6.0 at 90°C for 15 min. Mouse anti-human CD34 Class II monoclonal antibody , rabbit anti-human CD117 polyclonal antibody , and mouse anti-human myeloperoxidase (MPO) antibody  were incubated at 4°C over night. Horseradish peroxidase (HRP) labeled secondary antibody and the substrate diaminiobenzidine (DAB) were used according to the kit’s instructions. Then sections were viewed under the light microscope .P values ≤0.05 were considered statistically significant. All statistical analyses were conducted by using the SPSS 18.0 software program .Mann–Whitney U test was applied to compare the density of precursors between the relapse and no-relapse cases and their distance to endosteum in every cell group. Chi-square test and Fisher’s exact test were used for categorical data. A receiver operating characteristic curve (ROC) analysis was performed and the corresponding area under the curve (AUC) was calculated to determine the value of the precursor density in predicting a recurrence. Survival curves were constructed using the Kaplan–Meier method and the log-rank test was used to test the difference in RFS and OS between cases with different densities of ALIP-like clusters. RFS was defined as the time between diagnosis and the onset of the first relapse, and OS was defined as the time between diagnosis and the occurrence of death or when lost to follow-up. Cox proportional hazard regression was used to model the RFS. The data were described as mean ± standard deviation (SD), Ages of patients in the relapse group and no-relapse group were (51.85 ± 17.96) years and (49.86 ± 16.96) years respectively. The ratios of male/female in the two groups were 24/15 and 10/11 respectively . However, clustered precursors in relapse patients were significant higher than those in no-relapse patients (P = 0.0075). In ROC analysis of recurrent prediction, single, double and clustered precursors resulted in an AUC of 0.551, 0.573, and 0.711, respectively  and hematologic relapse were compared. As a result, the median time from histologic relapse to hematologic relapse was 8 months  and OS (P = 0.045) for cases with cluster density > 4.0/mm2.As shown in Figure y Figure B and wit2 was associated with higher relapse risk  or some benign disease with impaired BM, such as BM necrosis and stromal alterations in systemic lupus erythematosus patients , but notOur observations showed that single and double precursors were located near the endosteum, while ALIP-like clusters were in intertrabecular region. This is analogous with a recent study that showed that single normal hematopoietic stem cells (HSC) were located close to the endosteum. However, more mature precursors were located farther away from the endosteum and expanded to clusters . Likewis2 of ALIP-like cluster density in BM sections within hematologic remission is a strong independent prognostic factor for AML after achieving a complete remission, and consider it as an indication of histologic relapse and early interventions might be considered as a pre-requisite in histological relapse to improve eventual clinical outcomes. Here, we suggest BM biopsies should be considered while other minimal residual disease measurements such as reverse transcription-polymerase chain reaction (RT-PCR), flow cytometer, etc. are not available. Nonetheless, there are some limitations in this study. Firstly, cytogenetic and molecular marker assays are not routinely performed at this hospital, comparison and combination with other MRD measurements such as RT-PCR, flow cytometer, etc. [In this current study, we propose that at a value of greater than 4.0/mmer, etc. ,23 are l2, although in hematological response, urgent interference should be needed to prevent early recurrence and consequently improve the OS.Taken together, in this retrospective study, we found during CR of AML patients, except for single and double precursors, there existed clustered precursors of 3 ~ 5 cells. The clustered precursors are morphologically and anatomically analogous to ALIP in MDS. Similar with the prognostic value of ALIP in MDS, these ALIP-like clusters in BM sections during CR of AML could also be used as an independent predictor of early relapse and poor outcome for AML patients. Finally, we proposed that while ALIP-like cluster density increased to over the threshold of 4.0/mmThe authors declare that they have no competing interests.YY performed laboratory tests and collected patient data and analyzed the data. ZW analyzed the patient data and wrote the paper. JZ, YY, YZ, SL and HW performed the laboratory tests. JS provided patient data, analyzed the data and wrote the paper. All authors read and approved the final version of the submitted manuscript.


Thrombocytopenia is a very frequent disorder in the intensive care unit. Many etiologies should be searched, and therapeutic approaches differ according to these different causes. However, no guideline exists regarding optimum practices for these situations in critically ill patients. We present recommendations for the management of thrombocytopenia in intensive care unit, excluding pregnancy, developed by an expert group of the French-Language Society of Intensive Care (Société de Réanimation de Langue Française (SRLF), the French Language Group of Paediatric Intensive Care and Emergencies (GFRUP) and of the Haemostasis and Thrombosis Study Group (GEHT) of the French Society of Haematology (SFH). The recommendations cover six fields of application: definition, epidemiology, and prognosis; diagnostic approach; therapeutic aspects; thrombocytopenia and sepsis; iatrogenic thrombocytopenia, with a special focus on heparin-induced thrombocytopenia; and thrombotic microangiopathy. Thrombocytopenia is the hemostatic disorder that is most frequently encountered in intensive care and is seen in 41–66% of patients (close to 50% at admission). In view of the mortality associated with thrombocytopenia, the numerous associated pathological conditions seen in intensive care, and the lack of recommended treatment strategy, the SRLF has drawn up these recommendations.An SRLF expert group drew up recommendations, giving reasoned arguments for each of six fields of application defined by the organizing committee, including pediatric specificities. Each expert (or expert subgroup) then presented and justified the content and form of the proposed recommendations, changes to which could be made during the ensuing discussion. The revised recommendations were then put to the vote. The purpose was not to impose a single expert point of view for all the recommendations but to delineate clearly the points of agreement that underpin the recommendations, as well as points of disagreement or indecision that may form the basis of future work.Recommendations were scored according to an approach based on the RAND/UCLA Appropriateness Method. After the first round of voting for each recommendation (score 1 for complete disagreement or lack of any proof to 9 for complete agreement or conclusive evidence), the highest and lowest scores were eliminated, and the score was taken as the median of the remaining scores. The upper and lower bounds were then considered as “strong agreement” if they lay within the range 7–9 and “strong disagreement” in the range 1–3. Such scores were considered definitive. Recommendations whose scores were not within these two ranges were voted on again, following the same principle. After this second round of voting, the scores were taken as follows:– Median and bounds within range [7–9] = “strong agreement”– Median and bounds within range [1–3] = “strong disagreement”– Median within range [7–9] with lower bound < 7 = “weak agreement”– Median within range [1–3] with upper bound > 3 = “weak disagreement”– Median within range [4–6] = “uncertainty”http://www.gradeworkinggroup.org/links.htm), the originality of which stems primarily from three assertions. First, characterization of the type of study alone  is insufficient to indicate the level of proof of a study. Second, the risk/benefit ratio is taken into account. Third, the exact wording of guidelines carries clear implications for users: “should be done/recommended…” (strong agreement) or “should not be done/not recommended” (strong disagreement), “should probably be done/is possible” (weak agreement), or “should probably not be done/is possible not to do” (weak disagreement).The methodology used was inspired by the GRADE system  for rating clinical guidelines (9/L (strong agreement).1. Thrombocytopenia is defined by a platelet count (PC) < 150 × 10in vitro agglutination of platelets, by preparing a blood film, or, if necessary, using a blood sample collected in a citrate tube (strong agreement).2. In intensive care, pseudothrombocytopenia should be discounted using 9/L or a > 30% decrease in PC. It also can be done for a drop in PC, independently of these cutoffs, depending on the pathological context (strong agreement).3. An etiological investigation is required to determine the causes and underlying mechanisms of a PC < 100 × 10(weak agreement).4. A more than 30% drop in PC should probably be considered to be associated with a poor prognosis (strong agreement).5. Changes in PC over time  should be considered to be associated with the prognosis (strong agreement).6. The underlying mechanisms and etiologies of thrombocytopenia in intensive care are often multiple, with peripheral causes predominating (strong agreement).7. Sepsis is the main etiology of thrombocytopenia in intensive care and is usually associated with disseminated intravascular coagulation (DIC) and2) (strong agreement).8. Diagnosis of DIC should be based on both clinical and biochemical criteria. International Society on Thrombosis and Haemostasis (ISTH) and Japanese Association for Acute Medicine (JAAM) scores can be used to objectify DIC .9. The clinical context should suggest the etiology of thrombocytopenia in most cases in intensive care (strong agreement).10. In thrombocytopenia in intensive care, bone marrow biopsy should not be performed routinely but can be considered when there is no obvious etiology or when other cell lineages are affected (strong agreement).11. Screening for antiplatelet antibodies is unjustified in the initial diagnostic approach to thrombocytopenia (strong agreement).12. It is important to know which diagnoses of thrombocytopenia justify special investigations and prompt initiation of specific treatments   (strong agreement).13. In intensive care, bleeding severity can be graded with a 5-point scale used in the literature 9/L (weak agreement).14. For a similar PC, central thrombocytopenia probably involves a greater risk of bleeding than peripheral thrombocytopenia. Bleeding complications occur above all with central thrombocytopenia < 20 × 1015. The decision to treat thrombocytopenia should be based on the PC but also on the:• Presence of active bleeding ,• Mechanism of thrombocytopenia , • Etiology,• Risk of thrombosis,• Risk of hemorrhage , and(strong agreement).• Associated treatments (strong agreement).16. If platelet transfusion is necessary, pooled standard platelet units should be used, except for allo-immunized patients, those receiving treatment for blood disorders, and those with blood disorders, all of whom should be given aphaeresis platelet concentrate (single donor)11 platelets) per 7 kg body weight in adults and per 5 kg in children (strong agreement).17. The number of platelet units transfused should be prescribed depending on body weight: 1 platelet unit (0.5 to 0.7 × 10(strong agreement).18. ABO/Rh1 (D)-compatible platelets should be preferred for transfusion (strong agreement).19. There is no need to calculate the percent platelet recovery (weak agreement).20. Depending on the context and in the event of severe risk of hemorrhage, it is probably necessary to measure the PC in the hour following the end of platelet transfusion 9/L, the clinical situation and factors that may influence primary or secondary hemostasis (strong agreement).21. The risk of hemorrhage in thrombocytopenia should not be defined by the PC alone, but by a combination of PC < 50 × 109/L in central or peripheral thrombocytopenia (strong agreement).22. In intensive care, prophylactic platelet transfusion should not be routine when the PC > 20 × 109/L (weak agreement).23. In intensive care, prophylactic platelet transfusion should probably be done in central thrombocytopenia when the PC < 20 × 109/L (strong agreement).24. In intensive care, in the case of severe hemorrhage, platelet transfusion is recommended when the PC ≤ 50 × 109/L in the following situations:25. In intensive care, platelet transfusion is probably necessary when the PC ≤ 50 × 10– Severe sepsis, with risk of severe hemorrhage or use of anticoagulant,– Invasive procedure,(weak agreement).– Pre- or postsurgical setting 9/L: after surgery of the central nervous system, liver, eye, and large blood vessels; in patients with polytrauma (weak agreement).;26. In intensive care, platelet transfusion is probably necessary when the PC ≤ 100 × 10(strong agreement).27. In intensive care, prophylactic platelet transfusion is not recommended in the following situations:posttransfusion purpura, thrombotic thrombocytopenic purpura (TTP), catastrophic antiphospholipid syndrome, hemolytic-uremic syndrome (HUS), heparin-induced thrombocytopenia (HIT) (weak agreement).28. In intensive care, prophylactic platelet transfusion is probably not recommended in cases of DIC (strong agreement).29. In intensive care, the risk of hemorrhage  and the risk of thromboembolic events should be assessed before deciding to administer antiplatelet drugs, curative anticoagulant treatment, or preventive anticoagulant treatment to thrombocytopenic patients 9/L or there is a major risk of hemorrhage (strong agreement).30. Antithrombotic prophylaxis using unfractionated heparin (UFH) or low-molecular-weight heparin (LMWH) should probably be prescribed routinely in all adult patients admitted to intensive care, except when the PC < 30 × 10(strong agreement).31. Any interruption of antiplatelet drugs in an intensive care patient should be as short as possible, especially if the patient has a drug-eluting stent 9/L, other risk factors, etc.) (weak agreement).32. When a patient admitted to intensive care is already taking antiplatelet drugs, these should probably be withdrawn if there is a risk of hemorrhage (PC < 50 × 10(strong agreement).34. Use of immunoglobulins in thrombocytopenic patients with sepsis is not recommended (strong agreement).35. Thrombocytopenia is very frequent in infectious purpura fulminans, because DIC is more severe that in other septic shock etiologies in children (strong agreement).36. Apart from immediate parenteral administration of a first dose of antibiotic as soon as infectious purpura fulminans is suspected, there is no recommended treatment specific to infectious purpura fulminans compared with other septic shock etiologies in children(strong agreement).37. For infectious thrombocytopenia, routine administration of treatments specific to hemostasis  is not recommended because of the high risk of hemorrhage. It can be considered in the most severe cases, always following individualized reassessment of the risk/benefit ratio (strong agreement).38. Etiological diagnosis of drug-induced thrombocytopenia in intensive care should be guided by its origin  and mechanism (immune versus nonimmune) weak agreement).39. HIT should probably be suspected for any thrombocytopenia in intensive care ((strong agreement).40. Drug-induced thrombocytopenia should be treated by withdrawal of the drug concerned, which retrospectively confirms the diagnosis 9/L or with life-threatening bleeding) of drug-induced thrombocytopenia with an immune mechanism, intravenous immunoglobulins or even plasmapheresis is possible (weak agreement).41. In very severe forms (PC < 5 × 10(strong agreement).42. Thrombocytopenia after chemotherapy most often is linked to myelosuppression, its severity and duration depends on the type and dose of cytostatic drugs used and on the initial PC 9/L with signs of bleeding and should be routine for a PC < 10 × 109/L (strong agreement).43. In central thrombocytopenia after chemotherapy, platelet transfusion is recommended for a PC < 20 × 10(strong agreement).44. Severe thrombocytopenia in the multiparous woman or the patient who has received multiple transfusions is indicative of posttransfusion purpura, which can be treated with intravenous immunoglobulins (strong agreement).45. For patients at high risk of hemorrhage without liver failure, citrate should be the anticoagulant of choice for intermittent and continuous renal dialysis (strong agreement).46. Extracorporeal membrane oxygenation (ECMO) or extracorporeal life support (ECLS) in intensive care is always accompanied by a decrease in PC and requires anticoagulation, generally with low doses of heparin  for the first few days, with dosage adjustment according to hemostasis tests (strong agreement).47. Whichever heparin is used (UFH or LMWH), baseline PC should be measured on initiation or during the first 24 hours of treatment (weak agreement).48. PC should probably be measured twice in any patient exposed to UFH during the past 3 months, first upon initiation of heparin therapy (UFH or LMWH) and then the next day (strong agreement).49. In intensive care, PC should be measured twice a week during the first 3 weeks of heparin therapy (UFH or LMWH)(strong agreement).50. Warkentin 4-T score should be used to assess the probability of HIT, even though it has not been specifically validated in intensive care .51. In patients with a Warkentin 4-T score < 2, a laboratory test of HIT should probably not be done (weak agreement).52. Two types of diagnostic laboratory tests should probably be combined in any patient with suspected HIT: one functional and one immunoenzymatic (ELISA) to screen for antiheparin PF4 antibodies (strong agreement).53. Heparin should be excluded if there is a history of HIT. A nonheparin anticoagulant is recommended (strong agreement).54. If there is a history of HIT, danaparoid is the first-line anticoagulant in intensive care (weak agreement).55. In a patient with suspected HIT and a Warkentin 4-T score ≥ 2, withdrawal of heparin should probably be considered (strong agreement).56. In a patient with suspected HIT and a Warkentin 4-T score ≥ 4, heparin should be replaced immediately by an empirical nonheparin treatment at curative dosage: danaparoid sodium, lepirudin, or even bivalirudin (weak agreement).57. In intensive care, fondaparinux is probably not recommended as replacement therapy during the acute phase of HIT (strong agreement).58. In intensive care, the introduction of vitamin K antagonists during the acute phase of HIT is contraindicated (strong agreement).59. Thrombocytopenia associated with mechanical hemolytic anemia should suggest TMA, even in the absence of organ failure (strong agreement).60. In any patient with suspected TMA, the blood samples (2 dry tubes and 2 EDTA tubes) for etiological investigation  should be collected before any treatment, without delaying its initiation (strong agreement).61. The first-line treatment of TMA should be based on emergency plasma exchange (PE) (rate 60 mL/kg). If PE is not initially possible, plasma should be infused at 20 mL/kg when possible (strong agreement).62. In TMA, PE should be continued daily for at least 5 to 7 days until the PC normalizes and is stable for at least 48 hours. The rate of PE is lowered progressively, on a case-by-case basis (strong agreement).63. In adults and children with TMA, platelet transfusion is strictly contraindicated unless there is life-threatening bleeding (strong agreement).64. In adults with TMA, an invasive procedure is not an indication for routine prophylactic platelet transfusion because of potentially lethal adverse effects (weak agreement).65. In children with TTP, an invasive procedure is probably not an indication for routine prophylactic platelet transfusion because of potentially lethal adverse effects (strong agreement).66. In children with HUS, routine prophylactic platelet transfusion should be restricted to invasive procedures that have a high risk of hemorrhage (strong agreement).67. For the specific treatment of TMA, infusion of heparin, fibrinolytics, prostacyclin, or vitamin E is useless, even dangerous. There is no evidence for the efficacy of antiplatelet drugs (strong agreement).68. In TTP, patients should be routinely monitored and tested for myocardial ischemia (strong agreement).69. In TTP, methylprednisolone can be used if not contraindicated, in combination with PE (strong agreement).70. In congenital TTP, specific treatment should be based on administration of plasma (10 mL/kg every 2 to 4 weeks) E. coli (STEC) using culture on MacConkey agar and polymerase chain reaction to test for Shiga toxin (strong agreement).71. In HUS, stools or rectal swabs should be tested for Shiga toxin-producing (weak agreement).72. In HUS, if no STEC is found in the stools, the serum should probably be tested for anti-LPS IgM of the most frequent serogroups of STEC (strong agreement).73. Children with HUS should be transferred to a specialized department (nephrology or intensive care) for discussion of prompt initiation of hemodialysis (weak agreement).74. In adults and children with HUS caused by STEC, PE has not proven effective and should probably not be recommended (weak agreement).75. PE should probably be used in HUS caused by STEC with central neurological involvement Streptococcus pneumoniae, administration of plasma is probably contraindicated and only washed packed red blood cells or platelets should be used when the agglutination test is positive(weak agreement).76. In HUS caused by (strong agreement).77. First-line treatment of atypical HUS should be emergency PE (rate 60 mL/kg). If PE is not initially possible, plasma should be infused at 10–20 mL/kg when possible 78. If the response to first-line treatment is suboptimal, immunomodulators should be considered , in collaboration with a reference center. Suboptimal response is defined as follows: – Atypical HUS: failure to correct thrombocytopenia and/or to improve renal function at day 5 of standard treatment (strong agreement).– Acquired TTP: PC not doubled by day 5 of standard treatment or diseaseprogression (increased thrombocytopenia or onset of organ damage) duringintensive first-line treatment or on reducing the frequency of PE (strong agreement).79. In the absence of treatment, the prognosis of atypical HUS is guarded , particularly when there is factor H mutation  9/L is associated with an unfavorable prognosis.PC < 150 × 109/L.In intensive care, prophylactic platelet transfusion should be performed in cases of peripheral thrombocytopenia < 10 × 10E. coli ; TMA: thrombotic microangiopathy; TTP: thrombotic thrombocytopenic purpura; UFH: unfractionated heparin.DIC: disseminated intravascular coagulation; HIT: heparin-induced thrombocytopenia; HUS: hemolytic-uremic syndrome; LMWH: low-molecular-weight heparin; PC: platelet count; PE: plasma exchange; STEC: Shiga toxin-producing The authors and the experts declare that they have no competing interests.TV, BS, LD, LS, and PM contributed to the conception and design of the study, the draft, and critical revision of the manuscript, and all authors approved the final version.1. Organising Committee for the SRLF Recommendations and Assessments Board: M. Monchi, C. Bretonnière, T. Boulain, K. Chaoui, A. Cravoisy, D. Da Silva, M. Djibré, F. Fieux, D. Hurel, V. Lemiale, O. Lesieur, M. Lesny, P. Meyer, C. Milesi, B. Misset, D. Orlikowski, D. Osman, JP. Quenot, L. Soufir, T. Van der Linden, I. Verheyde (T. Van der Linden).2. Expert Coordinator (B. Souweine).3. Experts: N. Ajzemberg (Paris), P. Coppo (Paris), M. Darmon (Saint Etienne), S. Dauger (Paris), L. Drouet (Paris), I. Elalamy (Paris), B. François (Limoges), D. Gruson (Bordeaux), C. Loirat (Paris), B. Megarbane (Paris), F. Pene (Paris), B. Souweine (Clermont-Ferrand), F. Stephan (Marie Lannelongue), B. Tardy (Saint Etienne) (group of experts).Expert recommendations formalized under the aegis of the French-language Intensive Care Society , with the participation of the French Language Group of Paediatric Intensive Care and Emergencies (GFRUP) and of the Haemostasis and Thrombosis Study Group (GEHT) of the French Society of Haematology (SFH). Diagnostic algorithm for overt DIC according to the International Society on Thrombosis and Haemostasis (ISTH), from Taylor Jr FB et al Thromb Haemost 2001. Click here for file Diagnostic score for DIC according to the Japanese Association for Acute Medicine (JAAM). from Gando S. Crit Care Med 2006. Click here for file Clinical probability of HIT according to the Warkentin 4-T score. from Warkentin TE et al., Hematology (Am Soc Hematol Educ Program) 2003. Click here for file


Objective: The clinical relationship between brain-derived neurotrophic factor (BDNF) and cognitive function or mild cognitive impairment (MCI) is not well-understood. The purpose of this study was to identify the relationship between serum BDNF and cognitive function and MCI, and determine whether serum BDNF level might be a useful biomarker for assessing risk for MCI in older people.Materials and Methods: A total of 4463 individuals aged 65 years or older (mean age 72 years) participating in the study. We measured performance in a battery of neuropsychological and cognitive function tests; serum BDNF concentration.Results: Eight hundred twenty-seven participants (18.8%) had MCI. After adjustment for sex, age, education level, diabetes, and current smoking, serum BDNF was associated with poorer performance in the story memory, and digit symbol substitution task scores. Serum BDNF was marginally associated with the presence of MCI  when BDNF was 1.5 SD lower than the mean value standardized for sex and age, education level, diabetes, and current smoking.Conclusion: Low serum BDNF was associated with lower cognitive test scores and MCI. Future prospective studies should establish the discriminative value of serum BDNF for the risk of MCI. Mild cognitive impairment (MCI) is a transitional condition between normal cognitive function and a clinical diagnosis of probable Alzheimer’s disease (AD). MCI, including amnestic MCI, is a pathologically heterogeneous disorder in which many persons exhibiting mixed pathologies . Each individual was recruited from Obu, Japan, which is a residential suburb of Nagoya. To be included in this study, each participant was 65 years or older at the time of examination (2011 or 2012), resided in Obu city, and had not participated in another study. We excluded participants who had missing BDNF data and characteristics, diagnosed neurological disorders included stroke, Parkinson’s disease, AD, and depression, certified long-term care insurance, or functional decline of activities of daily living (ADL). Figure g. The collected serum was stored in polypropylene tubes at −80°C until assayed. BDNF concentrations were quantitatively determined by enzyme-linked immunosorbent assay (ELISA) using the DuoSet ELISA Development Kit from R&D Systems . Assays were performed using a specific human BDNF antibody ; no significant cross reactivity or interference was observed in this assay. Serum samples were diluted 1:50. Sample BDNF concentrations were then determined by non-linear regression from the standard curves. Measurements were performed in duplicate and averaged to give a value in picogram per milliliter, which was then expressed in nanogram per milliliter after correcting for sample dilution. “Low” and “High” concentration quality control pools were prepared by adding 10 or 100 ng to 5 ml portions of human serum , giving nominal concentrations of 2 and 20 ng/ml, respectively. The assays were performed by one laboratory . The repeatability of the BDNF ELISA, as measured by intra-assay precision was 3.8%, and the reproducibility, as measured by inter-assay precision, was 7.6%.Whole blood samples were collected from each patient by venipuncture. To obtain serum, whole blood samples were allowed to coagulate at room temperature (RT) for 30 min and then centrifuged at RT for 15 min at 1000 × We defined MCI based on previous studies  by one-way analysis of variance (ANOVA) in both sexes. A linear regression was used to analyze the relationships between BDNF concentration and age and education in both sexes. Participants were divided into two groups according to 1.0 or 1.5 SD from age- and sex-specific mean values among the four age-groups  participants who had BDNF levels below 1.0 SD and above 1.0 SD; and (b) participants who had BDNF levels below 1.5 SD and above 1.5 SD. Linear regression analyses (forced-entry) were used to reveal the relationships between BDNF concentration and cognitive performance. Multivariate logistic regression analyses, forced-entry, were used to determine adjusted odds ratios (ORs) and 95% confidence intervals (95% CIs), and to assess independent associations between the serum BDNF levels and MCI. The covariates of sex, age, and educational level, and significant variables in univariate analyses were added to the regression models to evaluate independent associations between BDNF and cognitive performances or MCI. Logistic regression models determined the crude OR and the adjusted OR of BDNF for 1.0 and 1.5 SD. Sensitivity, specificity, and positive and negative likelihood ratios of the BDNF values with MCI were calculated. We excluded the participants who scored ≤23 points on the MMSE and did not complain of memory loss. We used the data of MCI (n = 827) and cognitive healthy (n = 2533) elderly adults in the logistic regression analyses. All statistical comparisons were made at the 0.05 level of significance, and all data management and statistical computations were performed using the IBM SPSS Statistics 20.0 software package .Student’s t = 2.162, df = 4394, P = 0.031). BDNF concentrations declined with increasing age in both sexes   and women . Education level was associated with serum BDNF in women , but not in men .The mean BDNF concentrations were statistically significantly different in men (20.8 ± 5.6 ng/ml) and women .The comparison between participants who had BDNF levels below 1.0 SD and above 1.0 SD, revealed that the participants below 1.0 SD had a higher prevalence of diabetes, a lower proportion of smokers, higher scores of story memory, and a symbol digit substitution task, compared with participants who had BDNF levels above 1.0 SD. The results were similar for the comparison between the participants who had BDNF levels below 1.5 SD and above 1.5 SD. A comparison of MCI prevalence found no significant difference between the participants who had serum BDNF below and above 1.0 SD. In contrast, when serum BDNF was dichotomized according to 1.5 SD below the mean, a significant difference was found in MCI Table . The meat = 1.958, P < 0.05) and digit symbol substitution test scores . There was no significance between BDNF and MMSE for word-list memory, the tablet version of the Trail Making Test – Part A and B, or figure selection.Table In all, 827 participants (18.8%) had MCI. A total of 691 participants (15.5%) had BDNF levels below 1.0 SD from the mean, and 246 participants (5.5%) had levels below 1.5 SD from the mean. Table In our cross-sectional observational study of 4463 community-living older adults, serum BDNF was associated with a decline in story memory and digit symbol substitution test scores, even when adjusted for sex, age, education, diabetes, and current smoking. Moreover, serum BDNF levels of 1.5 SD lower than the age- and sex-adjusted means were associated with a significant risk of MCI. These results suggest that serum BDNF may be a useful biomarker of cognitive function and MCI status in the elderly.In demographic variables, serum BDNF was higher in women than men. Similar results were found by Trajkovska et al.  using boAmong lifestyle measures, diabetes and current smoking showed significant differences between the participants who had high and low serum BDNF levels. Low levels of BDNF accompanied impaired glucose metabolism. Krabbe et al. reported that plasma levels of BDNF were decreased in participants with type 2 diabetes independent of obesity . Further studies will be needed to establish the relationships between serum BDNF and executive function in the elderly adults.Brain-derived neurotrophic factor is highly concentrated in the hippocampus . The result suggests that it is necessary to review the discrimination point to screen MCI in the community with high sensitivity. BDNF is reduced in elderly individuals with major depression and bipolar disorder, with distinct dynamics according to the disease stages, treatment, or the presence of cognitive impairment (Molendijk et al., In conclusion, we provide preliminary evidence that serum BDNF can be associated with lower cognitive test scores in older people. In our cohort, serum BDNF was marginally associated with the presence of MCI when BDNF was 1.5 SD lower than the mean age- and sex-adjusted values. Future prospective studies should establish the discriminative value of serum BDNF for a risk of MCI and its validity as a screening test for this population.Study concept and design: Hiroyuki Shimada, Takao Suzuki; acquisition of data: Hyuma Makizako, Takehiko Doi, Daisuke Yoshida, Kota Tsutsumimoto, Yuya Anan, Kazuki Uemura; analysis and interpretation of data: Sangyoon Lee; critical revision of the manuscript: Hyuma Makizako, Hyuntae Park; statistical analysis: Hyuntae Park; drafting of the manuscript: Hiroyuki Shimada; obtaining funding: Hiroyuki Shimada, Takao Suzuki, Hyuma Makizako; study supervision: Takao Suzuki.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.