DNase-seq and ATAC-seq are broadly used methods to assay open chromatin regions genome-wide. The single nucleotide resolution of DNase-seq has been further exploited to infer transcription factor binding sites (TFBSs) in regulatory regions through footprinting. Recent studies have demonstrated the sequence bias of DNase I and its adverse effects on footprinting efficiency. However, footprinting and the impact of sequence bias have not been extensively studied for ATAC-seq.Here, we undertake a systematic comparison of the two methods and show that a modification to the ATAC-seq protocol increases its yield and its agreement with DNase-seq data from the same cell line. We demonstrate that the two methods have distinct sequence biases and correct for these protocol-specific biases when performing footprinting. Despite the differences in footprint shapes, the locations of the inferred footprints in ATAC-seq and DNase-seq are largely concordant. However, the protocol-specific sequence biases in conjunction with the sequence content of TFBSs impact the discrimination of footprint from the background, which leads to one method outperforming the other for some TFs. Finally, we address the depth required for reproducible identification of open chromatin regions and TF footprints.We demonstrate that the impact of bias correction on footprinting performance is greater for DNase-seq than for ATAC-seq and that DNase-seq footprinting leads to better performance. It is possible to infer concordant footprints by using replicates, highlighting the importance of reproducibility assessment. The results presented here provide an overview of the advantages and limitations of footprinting analyses using ATAC-seq and DNase-seq.The online version of this article (10.1186/s13059-019-1654-y) contains supplementary material, which is available to authorized users. These tissue- and developmental stage-specific regulatory elements reside in nucleosome-free, accessible regions of the genome that are hypersensitive to nuclease attack [The discovery and characterization of e attack . Digestie attack , 3 and we attack , 5 or the attack . A more e attack . Both prTranscription factors (TFs) bound at CREs are major regulators of gene expression . As protThe efforts to assay bound sites genome-wide via TF footprinting have come under scrutiny by studies demonstrating that DNase I cleaves the underlying DNA in a non-uniform manner, where sequence composition dictates the cleavage propensities  , 29. ThiRecent efforts have explored the feasibility of TF footprinting with ATAC-seq , 25, 30;Early ATAC-seq libraries generated with the original protocol have large numbers of reads mapping to mitochondrial DNA (mtDNA) that need to be discarded, which severely impact the final library depth . For an p value < 2.2e−16), with a slightly higher odds ratio for the modified protocol (13.15 vs. 10.75). This improved agreement at the open chromatin region level, albeit moderate, provided further support that avoiding detergent lysis increases the concordance between ATAC-seq and DNase-seq.To adequately quantify the protocol-related differences of ATAC-seq vs. DNase-seq, we also generated a single-hit DNase-seq library in K562 cells and compared this alongside three other publicly available single-hit DNase-seq datasets , a factor with a well-known, high information content binding site with substantial available ChIP-seq data including in HEK293 cells , see the “We next investigated the individual contributions of bias modeling and replicates to this increased concordance and accuracy. The contribution of the replicates comes from the application of IDR as mentioned above, which creates a systematic way to find relevant cutoffs for the footprint score. To elucidate the contribution of bias, we first trained CTCF footprint models in the combined ATAC-seq and DNase-seq replicates, as outlined above, but using a uniform background, which is equivalent to no bias correction see The “” sectionPrevious studies that inferred cell type-specific TF binding site annotations from DNase footprint data typically used very large datasets (with hundreds of millions of reads per cell type) , 17. To To elucidate whether the previous observations would also apply more generally beyond CTCF, we conducted the footprinting analysis on other factors. The limited availability of ChIP-seq data in HEK293 cells motivated an experimental setup to learn the footprint shapes in K562 cells, where ChIP-seq data is more abundant , for all TFs in both experimental settings, we calculated the area under the receiver operating characteristic curve (AUC), ranking candidate sites by FLR and considering those that overlap ChIP-seq peaks to be true binding sites. In order to assess how the performance is linked to the relationship between the footprint and background models, the Pearson correlations between these 2 models  for each TF were calculated and compared to the AUCs. The AUCs negatively correlated with the footprint-background model similarities in both ATAC-seq and DNase-seq datasets Fig. c, suggesAs the background component is derived directly from sequence bias and given our previous observation that DNase-seq bias correction shows a stronger positive effect compared to ATAC-seq bias correction, we once again explored the role of bias more explicitly. In particular, two of three factors for which ATAC-seq outperformed DNase-seq, MEF2A, and STAT1 had the lowest DNase I cleavage propensities  over their motif regions, among all assayed factors Fig. d, whereaDNase-seq has been widely used to assay open chromatin regions and TF footprints. The emergence and increasing use of ATAC-seq necessitate a systematic comparison of the two methods, especially for TF footprinting. Here, in a comparative setting, we have shown that although the two methods have distinct sequence biases and generate different footprint shapes for the same TF, the sites they identify as bound are largely in agreement. However, the sequence content of TFBSs combined with protocol-specific sequence biases impacts footprinting efficiency for some TFs, leading to larger differences for these factors and making one method preferable to the other.There are opposing views on the library depth required for TF footprinting. Whereas some studies require at least 200 million reads , others Although the sequence bias of DNase I is well characterized, there is still no consensus about the benefits of bias correction for TF footprinting. Whereas some studies report increased accuracy upon bias correction , others k-mer with eight meaningful positions is used to correct ATAC-seq data [Recent studies have proposed several Tn5 bias correction methods, and in order to rule out that this observation resulted from our 6-mer-based approach  and Dulbecco’s modified Eagle’s medium (DMEM), respectively, both complemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin.DNase-seq experiments were conducted on 50 million cells as previously described , with thhttp://broadinstitute.github.io/picard/). Further processing was performed to filter out contigs as well as the Y and mitochondrial chromosomes and retain only the reads that aligned concordantly as a pair within the expected fragment length range (38–1500 bp).ATAC-seq experiments were performed on 50,000 cells for the K562 samples and 100,000 cells for the HEK293 samples, following the published protocol  but incrLibrary complexity and saturation were calculated using the preseq program , using tIn order to find open chromatin regions, peak calling was performed on the processed DNase-seq and ATAC-seq datasets using JAMM , with paWhere replicates were available, peaks in agreement between the two replicates were found using the irreproducible discovery rate (IDR) pipeline . SpecifiFor K562 ATAC-seq datasets, where replicates were not available, reads of the modified dataset with no lysis buffer were randomly subsetted to match the library depth of the original protocol with 10 min lysis, and peaks were called using JAMM as described above, with the addition of the -e auto parameter for automatic estimation of a minimum fold enrichment. These K562 ATAC-seq peak sets were used to infer signal to noise ratios by calculating log2.The sequence bias of the Tn5 transposase was calculated in the form of 6-mers, similar to the previous calculations of DNase bias . To thisThe Tn5 dimer cleaves the plus and minus strands with a 9-bp offset, and consequently, most studies analyzing ATAC-seq datasets employ a correction where the reads that align to the plus strand are shifted by + 4, and the reads that align to the minus strand are shifted by − 5 bases, to update the read start sites to represent the center of this 9-bp core sequence. However, we focus on the actual 5′ ends of the reads, as these are the cleavage sites, akin to the DNase I cut sites. Our first reason for this is illustrated in Additional file The average Tn5 transposition propensity in a candidate binding site of a given transcription factor was calculated by retrieving and counting all 6-mers associated with the site (without flanks). The counts were multiplied by the Tn5 transposition propensities of the associated 6-mers, summed and normalized by the total number of 6-mers in the site. The same calculation was applied for DNase, using the previously calculated DNase cleavage propensities per 6-mer .−6), we found the corresponding TFBS scores and reported the one closest to the lowest score in each motif set and the associated FPR as an empirical p value. This demonstrated that all our sets included significant motif matches, with the lowest empirical p value being 5 × 10−5 , where each value corresponded to the cleavage/transposition probabilities at a given nucleotide. For the footprint component, these probabilities were found by computing the aggregate DNase or ATAC-seq signal (from the 5′ ends of the reads) around the TFBSs that overlap ChIP-seq peaks for that factor and re-estimating the signal via expectation maximization. For the background component, the probabilities were calculated as the signal that would be expected solely due to the protocol-specific bias values, given the sequences around the candidate TFBSs (parameter Background = “Seq”). As we had previously not observed a distinct difference in performance, the background was kept fixed and not re-estimated (parameter Fixed = T). Once both components were learned, footprint scores were calculated for all candidate TFBSs, as the log odds of footprint vs. background . To learn footprint models without bias correction, our method was applied as described above, but with a uniform, fixed background model that assumes equal cleavage probabilities at each nucleotide.Transcription factor footprinting was performed with a site-centric method from our lab as previously described . SpecifiThe IDR strategy was applied here as well where replicates were available, to find reproducible footprints. To this end, candidate TFBSs with positive FLRs in both replicates were chosen and ranked by FLR. IDR analysis was performed with the same parameters as explained for peak calling, where FLR values replaced signal intensities. Again, the number of sites that passed the stringent 0.01 IDR threshold was noted. Finally, TFBSs were ranked by the average FLR from the two replicates and truncated according to the IDR result. This led to the reproducible FLR-IDR footprints.Footprint model AUCs  were calculated by fourfold cross-validation. Briefly, the data was split into four parts, and for TFBSs in each part, FLR was calculated using footprint and background models learned from the other three parts. TFBSs were ranked by FLR, and those intersecting ChIP-seq peaks were labeled as the true positives. The AUCs obtained from the four parts were averaged to obtain the final value. Similarly, sensitivity and specificity measures were also obtained using models trained on three out of four parts of the data and tested on the remaining part.Correction of Tn5 sequence bias in K562 ATAC-seq data with the seqOutBias  softwareAdditional file 1:Figure S1. Read count correlations of all K562 datasets and comparison of K562 ATAC-seq datasets generated with the original (10 min lysis) and modified (no lysis buffer) protocols. Figure S2. Read count correlations of all HEK293 datasets. Figure S3. Analysis of reproducible peaks in HEK293 cells. Figure S4. Library complexity and saturation plots for HEK293 ATAC-seq datasets. Figure S5. Analysis of footprint models learned in HEK293 datasets and assessing the effects of bias correction on footprinting performance. Figure S6. The relevance of the learned footprint models as illustrated by model similarity across datasets and concordance between footprint scores and ChIP-seq signals. Figure S7. Analysis of NRF1 footprints. Figure S8. Method and TF-specific footprinting efficiency. Figure S9. 6-mer bias correction strategy for ATAC-seq datasets. Table S1. General statistics of the ATAC-seq datasets generated in the study. Table S2. Descriptions, accession codes and final read counts for the utilized DNase-seq datasets and libraries generated by Tn5 transposition of deproteinized genomic DNA. Table S3. Scheme for ATAC-seq library comparisons for JAMM-IDR peak calls or FLR-IDR footprint calls. Table S4. ChIP-seq peaks used in the analysis. Table S5. PWM IDs used for genome-wide motif searches. (PDF 3299 kb)


Tumour microenvironment, including tumour–stroma ratio (TSR), mighthelp identifying high-risk cancer patients. However, the significance of TSR ingastric cancer is unclear, especially in the intestinal and diffuse subtypes. Theaim of this study was to investigate the tumour–stroma ratio in gastricadenocarcinoma, and its intestinal and diffuse histological subtypes, in relationto prognosis.Five hundred and eighty-three gastric adenocarcinoma patients whounderwent surgery in Oulu University hospital during years 1983–2016 were includedin this retrospective cohort study. TSR was analysed from the slides that wereoriginally used for diagnostic purposes. Patients were divided into stroma-poor(≤50% stroma) and stroma-rich (>50% stroma) groups and TSR was analysed inrelation to 5-year mortality and overall mortality.Patients with stroma-rich tumours had worse 5-year prognosis  compared to stroma-poor tumours. Stratified analysisshowed that stroma-rich tumours had worse 5-year prognosis in both intestinal  and diffuse histological types compared to stroma-poor tumours, respectively.High proportion of stroma is an independent prognostic factor inboth intestinal and diffuse histological subtypes of gastricadenocarcinoma. Prognosis remains poor despitethe development in treatments during the last decades, with 5-year mortality of29%2.Gastric adenocarcinomas are classified with TNM classification based on depth ofinfiltration and number of local lymph node- and distal metastases. However, somepatients with early T-stage without lymph node- or distant metastases still havepoor outcomes. Therefore, it would be important to find new factors to recognisesuch high-risk patients that would benefit the most from aggressive adjuvanttherapies after surgery.Gastric cancer is the fifth most common malignancy and the third mostcommon cause of cancer-related death worldwide3. Cancer-associated fibroblasts (CAFs) that producethe components of desmoplastic stroma seem to be very important in thisregard4.The amount of stroma on haematoxylin–eosin (HE)-stained sections of a tumour can beeasily assessed without additional stainings, and its assessment is potentiallyclinically applicable. Tumour–stroma ratio (TSR) is defined as the proportion oftumour cells relative to surrounding stroma, and it has been recognised as apotential prognostic factor for various solid tumours5. Few previous studies withlimited sample size have suggested that gastric cancers with high proportion ofstroma (low TSR) have poorer prognosis compared to gastric cancers with lowproportion of stroma (high TSR)9. However, the relevance of TSR inthe different histological types of gastric adenocarcinoma is poorly known,especially in the Western populations.Stromal component of tumour has been proven to greatly impact thedevelopment of the tumourThe aim of this study was to evaluate the reproducibility of theassessment of TSR and to elucidate the association between TSR and prognosis ingastric adenocarcinoma and its histological subtypes in a Western cohort.This study was a retrospective cohort study in a single institutionin a tertiary care hospital in Northern Finland. There were 601 patients whounderwent gastrectomy for gastric cancer in Oulu University Hospital between years1983 and 2016. Five hundred and eighty-three of those were gastric adenocarcinomapatients whose diagnostic tissue sections were available for analysis and wereincluded in the study.10. The immutable national personal numbersassigned to each resident in the country were used to combine data from thepatient records and the 100% complete follow-up data from the Causes of DeathRegistry at the Statistics Finland. Follow-up data were available until the end of2016.The patients were identified from the archives of the Department ofPathology at the Oulu University Hospital, Finland. Clinical data for each patientwere obtained from patient records, including operation charts and pathologyreports. For each patient, the HE-stained slides originally used for diagnosticpurposes were retrieved from the pathology archive and reviewed by theinvestigators, and those cases that were not classified by Lauren classificationor were classified as mixed, were additionally reviewed and classified by a singleexpert gastrointestinal pathologist (T.J.K). Cancers of intestinal histologicaltype were also graded for differentiation grade according to the WHO TNMclassification, seventh editionThe original, prospectively collected HE-stained diagnostic samplesused for clinical decision-making were used for the present study. MultipleHE-stained sections from each patient were viewed with a light microscope, and arepresentative section with deepest invasion was used for further analysis.Sections were scanned and digitised using Aperio AT2 , and TSR was analysed from scanned slides using an Aperio ImageScope bytwo independent researchers (N.K. and M.E.) blinded to the clinical and outcomedata.7.The part of tumour with most stroma was identified with a low magnification. Single vision field at ×100 total magnification wasused for analysis, and it was ensured that there were tumour cells present at allfour corners of the vision field. Smooth muscle, mucin and necrosis were excludedfrom analysis, and the remaining area, consisting of tumour cells and stroma, wasused. The area of stroma compared to the whole area under analysis was estimatedand scored by 10% intervals. For example, a score of 20% meant that 20% of theanalysed area consisted of stroma and 80% of the area consisted of tumour cells.As in the previously published literature, 50% ratio was defined as cut of point,and patients were divided into stroma-poor (proportion of stroma ≤50%) andstroma-rich (proportion of stroma >50%) groups7. When estimates differed by over 20% or were onthe different sides of the 50% cut-off point, the slide was re-assessed with athird investigator (T.J.K.), an expert gastrointestinal pathologist, and consensuswas reached.TSR was analysed from invasive parts of tumour where most stromawas present, as areas with a high amount of stroma were considered to be decisivefor prognosis based on previous resultsPrimary outcome of the study was 5-year all-cause mortality, andthe secondary outcome was overall all-cause mortality.χ2-test was usedto obtain p-values when comparing categoricalvariables. Continuous variables were compared, and p-values were obtained by T-test.The Kaplan–Meier method was used to compare mortality between groups, and thelog-rank test was used to determine statistical significance of differencesbetween groups. The Cox regression model was used to perform multivariableanalysis, providing hazard ratios (HR) with 95% confidence intervals (CI). Coxregression was adjusted for confounding variables: (1) year of surgery (<2000or ≥2000), (2) age at diagnosis (continuous variable), (3) sex ,(4) administration of preoperative chemotherapy (yes or no), (5) tumour stage(stage I–II or stage III–IV), (6) Lauren classification  and (7) radical resection (R0 orR1/2). Subgroup analyses were performed in Laurénintestinal, and diffuse-type gastric adenocarcinomas separately, adjusted forother confounders listed above, and also for histological grade (I–II or III) inthe intestinal type subgroup. Sensitivity analysis was performed excludingpatients with non-radical (R1/2) resection or distantmetastases. The point estimates in the sensitivity analysis did not differ fromthe main analysis and therefore only the results of the main analysis arepresented. IBM SPSS Statistics 24.0  was used for allstatistical analyses.Cohen’s kappa was calculated to analyse interobserver agreement.0 resection and 146 (25.0%) hadR1/2 resection. The patients withR1/2 resection included patients with non-curativeintent, as well as 34 (5.8%) patients who had distant metastases at the time ofsurgery. Median follow-up time was 26 months (range 0–396 months).A total of 583 patients diagnosed with gastric carcinoma andundergoing surgical resection were included in the study. Median age of thepatients was 69 years , with 352(60.4%) of patients being men and 231 (39.6%) women. Only 22 (3.8%) of patientsunderwent preoperative chemotherapy. Of 583 patients, 437 (75.0%) underwentmicroscopically confirmed RTSR was successfully analysed for each patient and the patientswere divided into stroma-poor (proportion of stroma ≤50%) and stroma-rich(proportion of stroma >50%) groups  of the patients,while 342 (58.7%) had stroma-rich tumours. In the stroma-rich group, there wereproportionally more patients with surgery after the year 2000, younger age atdiagnosis, high TNM stage, diffuse histological type and unradical resection. In the univariable analysis, the stroma-rich group had significantlyworse prognosis compared to the stroma-poor group  compared to the stroma-rich group .Similarly, in the multivariable analysis, the stroma-rich group had significantlyhigher 5-year mortality compared to the stroma-poor group . In multivariable analysis of thepatients with diffuse-type histology, the stroma-rich group had significantlyworse prognosis compared to the patients with stroma-poor tumours  compared to thestroma-poor group  reported an HR of 1.91,with 95% CI 1.43–2.56, for the risk of death in the stroma-rich group of, but thestudy included only patients with complete histopathological and survival dataavailable, making the selection bias apparent9. Another Chinese study (n = 225) suggested an HR of 2.75, with 95% CI of1.86–4.07, for the 5-year mortality in the stroma-rich group in univariableanalysis, but no adjusted analysis was presented for TSR, and the follow-up wasbased on telephone, mail and outpatient service7. A Korean study with 175 gastricsignet-ring cell carcinomas showed an HR of 2.50 with 95% CI of 1.48–4.25 for 5-yearsurvival in the stroma-rich group6. The only previous European study suggested anadjusted hazard ratio of 5.50, with 95% CI 2.34–12.92, but the study was conductedin a small (n = 106) and selected patientpopulation, and the analysis was not adjusted, for example, for age at diagnosis,sex or histological subtype8. Compared to the previous studies, smaller hazardratios for mortality comparing stroma-rich and stroma-poor groups were found in themultivariable analysis in the present study. These differences may be explained bynumber of factors. Three of the previous studies are fromAsia9, where the aetiology of gastric cancer is differentcompared to the Western countries, with for example higher prevalence of Helicobacter pylori in thepopulation11. In addition to differences in selection bias anddemographics, the large proportion of diffuse-type tumours, and the inclusion ofpatients with R1–2 resection and distant metastases in the present study could alsoexplain some of the differences in the estimates compared to the previous studies.However, the effect of R1–2 resection and metastasis on the results is not supportedby the fact that results of the sensitivity analysis did not differ from the mainanalysis, and that the inclusion of patients with R1–2 resection and metastasesshould rather inflate the associations to mortality, not mitigate them. The subgroupanalysis suggested that TSR is an independent prognostic factor for 5-year andoverall mortality in both intestinal and diffuse types of gastricadenocarcinoma.High proportion of stroma was associated with high 5-year mortalityand high overall mortality, which is in line with the four previous studies on TSRand gastric cancer12.CAFs have been found to mediate drug resistance of cancer13, promoteangiogenesis14 and induce tumour cells to undergoepithelial–mesenchymal transition, which enhances invasive and metastaticcapabilities of tumour4. They also contribute to immune evasion by tumourcells at least in some cancer types16.Accordingly, as stroma-rich tumours have more CAFs, they could reasonably benefitmore of the growth supporting microenvironment CAFs create.Currently it is not known why patients with higher proportion ofstroma experience worse outcomes than those with low proportion of stroma. Probablemechanisms are based on interactions with tumour and stromal cells that have beensubject to intense research in recent years17. Human assessment showed highinterobserver agreement in the present study, but it would be useful to examine andvalidate computer-based assessment methods for gastric cancer in the future studies.Patients with low TSR might benefit from intensification of postoperative treatment,for example, chemoradiotherapy. Further typing of desmoplastic stroma on HE-stainedclasses has also been shown to distinguish high-risk patients in colorectalcancer19. It would be interesting tostudy its applicability in gastric adenocarcinoma, and whether taking differentdegrees of stromal maturation into account could increase the prognostic power ofTSR. Prospective studies are needed to get more precise estimates of the prognosticvalue of TSR in gastric adenocarcinoma and its histological subgroups.The results of the present study have clinical and research relatedimplications. Our study provides further evidence on the prognostic value of TSR forgastric adenocarcinoma, also in the different histological subtypes of thismalignancy. The analysis of TSR can be easily replicated and routinely analysed fromHE-stained slides without additional immunohistochemistry or costs, making it easilyapplicable to clinical decision-making. Computer-assisted analysis for analysing theamount of stroma can be used, for example, in breast cancerIn conclusion, the results of this large study suggest that highproportion of stroma (low TSR) associates to poor prognosis in gastricadenocarcinoma, but its prognostic value might be lower than previously reported.High proportion of stroma is an independent prognostic factor in both intestinal anddiffuse histological subtypes of gastric adenocarcinoma.Supplementary table 1Supplementary figure 1Supplementary figure legend


Hypertriglyceridaemia is one of the most common causes of severe acute pancreatitis (SAP). However, the association between elevated triglycerides (TG) level in hospital stay and outcome in SAP patients with normal TG level at admission has not been clearly demonstrated. This retrospective study assessed the serum TG levels of patients with SAP admitted to the intensive care unit (ICU) in 2017. Variables with a statistically significant association with the incidence of in-hospital TG elevation, as determined by univariate analysis, were analysed using a logistic regression model to predictors. Of the 99 patients included in the study, TG levels were within the normal range in 59 (59.59%) patients at admission. Among patients with normal TG level when admitted to ICU, 28 (47.46%) experienced at least one episode of TG level elevation during their ICU stay. Elevated TG level in hospitalization is associated with an increased length of ICU stay, as well as increased mortality. In addition to other factors, propofol usage was independently associated with the occurrence of in-hospital-TG elevation. To conclude, we retrospectively investigated the incidence, outcome, and risk factors for in-hospital TG elevation events in SAP patients admitted to the ICU. We found a high incidence of both preexisting and in-hospital-acquired TG elevation in SAP patients admitted to the ICU. The TG elevation that occurred during the ICU stay was associated with worse outcomes and long-term hospitalization of the ICU. Propofol usage was independently associated with the TG elevation occurrence in the ICU. Acute pancreatitis, which is sudden inflammation of the pancreas, is usually a mild disease with a favourable outcome , 2. AbouThe inflammatory reaction that occurs in SAP is thought to be due to premature activation of pancreatic proteases, which promote acinar cell apoptosis and necrosis . PancreaThis single-centre retrospective study was conducted in the Department of Critical Care Medicine, West China Hospital, Sichuan, China. The clinical research ethics boards of the West China Hospital approved the study and waived the need for participants’ informed consent because of the study’s retrospective, anonymous, and non-interventional nature. All methods were performed in accordance with the relevant guidelines and regulations. We retrospectively analysed data via individual chart review from all patients admitted with pancreatitis to the Department of Critical Care Medicine between January 2017 to December 2017 using a prospectively collected hospital database. A specialised system  was used to ensure data integrity.The diagnosis of acute pancreatitis is based on symptoms, physical examination and laboratory selection values and requires two of the following three characteristics: 1) acute onset of upper abdominal pain, 2) serum amylase or lipase activity greater than 3 times the normal level, and 3) findings on cross-sectional abdominal imaging consistent with acute pancreatitis . All patGallstone SAP was diagnosed if imaging studies  showed gallstones or choledocholithiasis. Hyperlipidemic SAP was defined as TG levels greater than 11.3 mmol/L or TG levels greater than 5.65 mmol/L with grossly lipaemic serum. Alcoholic SAP was diagnosed if the patient had a history of heavy alcohol consumption before the SAP onset and a history of alcohol consumption of over 50 g/d for longer than 5 years.For the purposes of this study, TG elevation event was defined when a serum TG level > 1.70 mmol/L was detected. The patient population was divided into two groups based on serum TG level at admission: a normal TG level group (N-TG group) and an elevated TG level group (E-TG group). Serum TG levels during the ICU stay were reviewed to rule out any episodes of new-onset TG elevation among patients with a normal TG level when admitted to ICU. Patients in the N-TG group were included in the in-hospital TG elevation group (H-E-TG group) if they experienced any episode of TG elevation while hospitalised, whereas members of in-hospital normal TG level group (H-N-TG group) did not experience any TG elevation episodes.Venous blood for all laboratory tests (including TG) was drawn within 6 h after admission and again whenever required, based on the judgement of the physician. Arterial blood was collected for air blood gas analysis whenever necessary. Specifically, TG was measured at the Department of Laboratory Medicines of West China Hospital using a Biochemical Analyzer . Other laboratory parameters were collected at admission and during hospitalisation, including: prothrombin time, aspartate aminotransferase, total protein, red blood cell count, haemoglobin level, direct bilirubin, arterial carbon dioxide partial pressure, haematocrit, activated partial thromboplastin time, mean corpuscular volume, mean corpuscular haemoglobin, high-density lipoprotein cholesterol, fibrinogen, mean corpuscular haemoglobin concentration, red blood cell distribution width, platelet count, white blood cell count, international normalized ratio, thrombin time, arterial oxygen partial pressure, base excess, lactate, procalcitonin, total bilirubin, alanine aminotransferase, albumin, glucose, cystatin C, gamma-glutamyl transferase, low-density lipoprotein cholesterol alkaline phosphatase and TG. Data routinely recorded on admission included age, gender, aetiology, and disease severity using the Acute Physiology and Chronic Health Evaluation II (APACHE II) score and Ranson’s score at admission.The data were analysed using SPSS Statistics 17  and GraphPad Prism  software. Continuous variables are presented as means ± standard deviation or median and interquartile range, while categorical variables are expressed as percentages. Analysis of variance (ANOVA) tests were used to compare means for continuous variables. Fisher's exact test was used for categorical variables. Chi-square tests were used to compare proportions for categorical variables. The log-rank test was used to evaluate statistical differences in survival curves, which were constructed using the Kaplan-Meier method. Univariate and multivariate logistic regression analyses were used to identify variables predictive of TG elevation event. A p value of <0.05 indicated a significant difference.We identified 255 adult ICU patients with a diagnosis of pancreatitis during the study period. Twenty-one patients were excluded because they were readmitted to the ICU during the study period. One hundred and twenty-nine patients who met the diagnosis of acute pancreatitis were enrolled after patients who were admitted because of recurrent chronic pancreatitis were excluded. Moreover, 15 whose ICU stay was < 24 h and 11 who had only mild acute pancreatitis were excluded, as well as two who were aged < 18 years at admission and two with insufficient data. Thus, 99 patients admitted for SAP were ultimately included in the study. Serum TG at the time of admission was higher than 1.70 mmol/L in 40 patients, who were therefore categorised as E-TG group. Thirty-one patients with normal TG level at admission experienced at least one episode of TG elevation during their ICU stay, and these patients were included in the H-E-TG group .The baseline characteristics of the study population are summarised in In total, 40.40% (40 out of 99) of the patients with SAP had an elevated TG level at the time of ICU admission . No signIn-hospital TG elevation events occurred in 47.46% (28/59) of patients in the cohort . The serTo determine why patients with normal TG level at admission developed new-onset TG elevation events, we analysed treatments that may have induced TG elevation during the ICU stay. The principal findings of this study were that TG elevation event was common in patients admitted to the ICU with a diagnosis of SAP. Hospital-acquired new-onset TG elevation event, was associated with worse outcomes and a longer ICU length of stay. Risk factor analysis indicated that the amount of propofol usage is an independent risk factor for hospital-acquired TG elevation in patients with normal TG level at admission. As far as we know, this is the first study that studies the epidemiology of TG elevation acquired in the hospital and its possible impact on the adverse outcomes of a SAP patients group.The pancreas is a secretory organ with both endocrine and exocrine functions that is directly involved in lipid metabolism. The presence of free fatty acids in the duodenum causes the release of the peptide hormone pancreazymin-cholecystokinin, which induces the gallbladder to release bile salts and the pancreas to release pancreatic lipase , 14. PanOur study characterised demographic data associated with elevated TG level at admission and acquired at ICU stay in patients with SAP. Patients with TG elevation when admitted to ICU had higher severity scores and were younger than patients those without. Thrombocytopenia, electrolyte disorders, and high creatinine levels were highly prevalent in patients with TG elevation at admission. While, patients with hospital-acquired TG elevation had a higher Ranson’s score and were older than those with TG elevation at admission.An elevated TG level at admission was not associated with increased mortality in our cohort, which is in accord with the findings of a recent meta-analysis . In addiWe found that hospital-acquired new-onset TG elevation is associated with mortality. All the patients in N-TG group are non-hyperlipidaemia-induced pancreatitis which ensure the comparability of the study. The elevated TG levels observed in SAP patients with a normal TG level at admission could occur for several reasons. Establishing enteral nutrition in these patients is relatively difficult because of persistent intestinal dysfunction, as well as SAP-related complications such as gastrointestinal fistula. Inadequate evaluation of lipid needs during parenteral nutrition may result in TG elevation. Moreover, current guidelines recommend maintaining TG values below 12 mmol/L instead of within the normal range, so clinicians may be poorly informed about lipid control. In addition, accurately determining an individual patient’s need for lipids and proteins is extremely difficult in current clinical practice. Another reason for the occurrence of hospital-acquired TG elevation was prolonged sedation with propofol in patients with SAP who underwent mechanical ventilation. Our study indicates that propofol, not parenteral nutrition, was the main cause of hospital-acquired new-onset TG elevation. These results suggest that midazolam should be used instead of propofol to sedate patients with SAP, to avoid disturbing lipid metabolism . Future In our study, we defined TG elevation as a serum TG level greater than 1.70 mmol/L. Indeed, TG elevation is usually a biochemical diagnosis that is made on the basis of whether the fasting plasma TG concentration is above a certain cut-off point . ProposeThis study has some limitations which have to be pointed out. First, this study was retrospective and observation bias is a concern although data were from a prospectively collected database. Second, we only investigated the serum TG levels for 28 days after ICU admission, and we did not have TG records for all patients in our hospital database. We did not include the mean weight/BMI in the demographics as this may also influence the TG levels in patients. The lack of follow-up data prevents our analysis of out-of-hospital outcomes.To conclude, we retrospectively investigated the incidence, outcome, and risk factors for TG elevation in SAP patients admitted to the ICU. We found that a high incidence of hospital-acquired TG elevation in SAP patients admitted to the ICU. The TG elevation acquired during the ICU stay was associated with worse outcomes and long-term hospitalization of the ICU. Propofol usage was independently associated with the TG elevation occurrence in the ICU.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S1 FigNo significant difference in mortality was observed between E-TG group patients and N-TG group patients.(EMF)Click here for additional data file.S2 FigA significant difference in mortality was observed between H-E-TG patients and H-N-TG patients.(EMF)Click here for additional data file.S1 Minimal dataset(ZIP)Click here for additional data file.


The nanomaterial-integrated chitinous polymers have promoted the technological advancements in personal health care apparatus, particularly for enzyme-based devices like the glucometer. Chitin and chitosan, being natural biopolymers, have attracted great attention in the field of biocatalysts engineering. Their remarkable tunable properties have been explored for enhancing enzyme performance and biosensor advancements. Currently, incorporation of nanomaterials in chitin and chitosan-based biosensors are also widely exploited for enzyme stability and interference-free detection. Therefore, in this review, we focus on various innovative multi-faceted strategies used for the fabrication of biological assemblies using chitinous biomaterial interface. We aim to summarize the current development on chitin/chitosan and their nano-architecture scaffolds for interdisciplinary biosensor research, especially for analytes like glucose. This review article will be useful for understanding the overall multifunctional aspects and progress of chitin and chitosan-based polysaccharides in the food, biomedical, pharmaceutical, environmental, and other diverse applications. Chitin is the second most abundant natural structural polysaccharide after cellulose which is derived from exoskeletons of crustaceans, cell walls of fungi and insects ,2,3,4,5.In this review, we focus on various innovative strategies used for the fabrication of biological assemblies using chitinous-biomaterial interface for glucose sensors and their applications in food, biomedical and other areas. Different roles of CS/CT such as a protective polymer, immobilization matrix, interface modulators and transducer-amplifiers for biosensing devices will be discussed. The literature studies are categorized into: Blend or composites of CT/CS, CT/CS nanocomposites and the techniques for designing the interface based on enzyme functionalization and their influence on sensor communication. The application for glucose detection using glucose oxidase (GOx) in the biomedical field, food industry, environment, and other sections will be discussed . This reThe polymer composite consisting of two or more chemically and physically different materials is considered as a wonder material because it can yield a distinct new interface with structural and functional superiority ,25. AlthNanocomposites are composed of reinforced nano-sized materials in a dispersed polymer matrix, typically an organic matrix comprising inorganic nanoparticles/structures within . Nanomat2O2), then cross-linking is followed by the treatment of tripolyphosphate (TPP) [Nano derivatives of crustacean materials can be prepared by several methods, depending upon the source of chitin. For example, chitin nanowhiskers are commonly produced by hydrochloric acid hydrolysis , precipite (TPP) ,54,55,56te (TPP) .Chitinous nanocomposites have great potential for biomedical, pharmaceutical and other versatile applications. The desired properties of these engineered materials such as non-toxicity, biocompatibility, biodegradability, and low-allergenicity led to an increased interest in the use of implantable biosensors for continuous monitoring of physiological biomarkers. Due to superior physical properties including film-forming tendency, high surface area, porosity, tensile strength and conductivity, they can be easily molded into functional interfaces . Enzyme 2O2 produced from glucose [−7 to 3 × 10−5 mol·dm−3 [A biopolymer like CT offers remarkable functional and divergent biological properties based on electrostatic interaction with other materials. The protonation of the acetylamide group of CT helps enzyme immobilization due to its negative charge. An electrochemical sensor for glucose determination in a sport drink was developed using a modified carbon paste electrode (CPE). A thin film of immobilized GOx-CT-platinum (Pt) powder was developed at the CPE surface for detecting H glucose . The sammol·dm−3 . The effκ-, ι-, λ-) and CS were prepared for micro-encapsulation to protect GOx under acidic conditions [The CS also has many promising biocompatibility characteristics, including excellent adhesion property, high mechanical strength, and tunable functional groups for chemical modifications ,61. The nditions .Cryogel is a form of a gel formed by freeze-drying a sol. The porous structure of cryogel provides a large surface area for biomolecule immobilization, which also has a significant impact on enzyme activity and reusability ,72,73. TCommercial glucose sensors for daily blood sugar detection is a great breakthrough. However, these point-of-care devices do suffer from high cost due to one-time use, low sensitivity, and interference problems. Interference from other electroactive species in physiological sample is the main challenge for these sensors. Therefore, tremendous efforts have been dedicated to establishing an interference-free/resistant interface for these biosensing devices. The bioelectronics interface may advance the transducer’s efficiency though it’s the inherent selectivity or specificity to amplify the analytical signal. Therefore, an important thing is to achieve the best biochemistry between the interface matrix and enzyme, which also comply with the morphological demands of these fragile biological entities . The ideTodarodes pacificus were produced by ball milling technique, they showed high affinity and good biocompatibility for GOx [2 with high surface area and good electrical conductivity were synthesized by electrospinning and further polymerized with polyaniline (PANI). These polymerized nanofibers were optimized for GOx and HRP conjugation with CS [Chitosan is known to be a perfect material for GOx immobilization as it provides a protective shield to the fragile enzyme molecules. The amino group of CS makes it more suitable for enzyme immobilization as compared to the other polymers. Chitosan accommodates the three dimensional (3-D) conformation of GOx by providing comfortable hydrophilic vicinity and structural stability. CS also provides pseudo cellular conditions via the cross-linked network of polymeric chains which directly or indirectly maintains the enzyme-friendly microenvironment. For example, electropolymers such as poly(N-methypyrrol) (PNMP) were used to synthesize film at the PtE surface to fabricate a reusable glucose biosensor. CS-GA-GOx was dip-coated to prepare a second film on the PNMP film on the electrode. The leakage of the enzyme was prevented by cross-linking immobilization which was confirmed by 20 cycles of reuses with retention of 91.3% of initial current response . Sol–gel for GOx . In this for GOx . In anot for GOx . Gao et  for GOx . CS also with CS . This MP with CS . In anot with CS . Liposom with CS . Thus, vEnzymes are conjugated with nanomaterials and chitinous matrix to fabricate biosensors. The retention of the morphological features and biological activity are the key for high sensitivity, wide linear range, low LOD, and reproducibility of the devices. Skilled modification and linking of such biomaterial-based interfaces increase electric communication and speedup the reaction rate which ultimately enhance the sensor’s performance. As an example, the following section will discuss various innovative approaches and methodologies for the preparation of probes for glucose sensing.2O2 produced. However, these sensors are highly susceptible for the interference caused by the endogenous electroactive species and fluctuations in the oxygen tension [The first-generation glucose biosensors were based on the use of natural oxygen substrate and the detection of the H tension ,104,105.In most of the electrochemical biosensor, GOx-flavin adenine dinucleotide (FAD) redox center catalyzes the electron transfer from glucose to gluconlactone. The communication between GOx-FAD and electrode via direct electron transfer (DET) is a big challenge, especially for the third generation i.e., label-free glucose biosensors. It is well understood that an electron transfer of GOx is controlled mainly by reorganization energies; potential differences and orientations of involved redox active sites and distances between redox-active sites and mediator ,94. The The surface energy, shape and size of the nanomaterials directly and dramatically improve their conjugation with enzymes, and interface performance and pattern. As shown in 2O2 reduction mediated by PB-G at string surface.Self-assembled monolayers (SAMs) or layer-by-layer (LBL) structures often hinder the electron and substrate-product transportation due to compact architecture. Being biodegradable, non-toxic. and highly bio-compatible, CS composites are proved to be the best biorecognition transducer. Some studies investigated the application of chitosan nanocomposites as an electrode matrix/material for glucose sensing where minimum interference at low potential is desired . The incTodarodes pacificus (GCSPs) with porous structure effectively promoted the immobilization of enzyme by providing comparatively larger conductive surface and more active sites for the GOx molecules [A 3-D porous film of CS and single-walled carbon nanotubes (SWCNTs) were utilized to construct a thin film-electrode after entrapping and cross-linking with GOx. The enzyme immobilized on this CS-SWCNTs film had higher enzyme loading and enzyme activity as compared to the non-porous planar films . Such elolecules . Besidesolecules . In thisks) of the GOx in CS-GR nanocomposite suggested that the modified electrode provides direct electron transfer between the redox center of the enzyme and the electrode surface due to CS-nanostructured confinement [Graphene (GR) is a two-dimensional monolayer of carbon atoms bonding with sp2 hybrid orbitals . Qian anfinement .A single nanofiber electrode made up of one-dimensional (1D) mesoporous ZnO/CS inorganic–organic hybrid nanostructure with high enzyme loading and stability features was reported  Figure .This device had mesoporous nanostructure with protuberances which favored enzymes loading via electrostatic adsorption and enhanced electrical communication efficiency. This free probe type biosensor prototype is highly suitable for micro-targets detection in microcell and enzymatic studies. It also has the potential to be inserted into single cell or other microorganism for biological studies in the future.Most of the electrochemical biosensor are based on the enzyme-catalyzed reactions and electron transfer between enzyme(s) and electroactive species with electrodes/transducers. However, one of the big challenges is to DET at the surface of bare working electrode (for example: GCE disc electrode). The insulation-shelled redox center of the bare electrode surface directly hamper the performance of the sensor . TherefoChitosan possesses excellent adhesive film forming property, it has been demonstrated to be the most suitable matrix for inclusion of single or multiple nanostructures via coating techniques. CS sol-gel to form film or membrane, are the first choice to construct an enzyme-electrodes. CS-hydrogel with or without other constituents showed the pH-dependent volume phase transition which was highly useful and effective for enzyme-nanomaterial integration in film layout. This strategy is also convenient for holding its natural properties to construct GOx and other analytical important sensors. Herein, we have discussed some of the examples where different configuration methodologies involving CT/CS films for probe modification to prepare enzyme-electrode interface and their application for either reusable or disposable sensors.2O2. New CS-nanocomposite matrix comprising polypyrrole nanotubes decorated with AuNPs was prepared by simple physical entrapment method [The 3-D network of CS can be formed by addition of sequester biomolecules/nanomaterials and this thick, uniform sol-gel can be drop cast on the tip/surface of the electrode to form film structure after drying. The porous and adhesive feature of the sol-gel CS is an effective entrapment/encapsulation matrix for the immobilization of biological element such as GOx. The electrode could also be modified by blank CS film formed at the top of the working electrode first, and then enzyme was linked via chemical treatment. Nafion (Nf), butyl carbitol acetate, dimethylformamide, cellulose, cellulose acetate, polyvinyl alcohol, etc. have been used as CS-film binder at electrode surface . Strong t method . The elet method . The phyt method .2 was proved for amperometric glucose detection. The excellent stability for 50 days with 90% enzyme activity could be attributed to two features. First, as the enzyme was physically entrapped in the CS-SiO2 gel, large quantities of hydroxyl and amino groups help to maintain the enzyme activity by forming strong interaction between GOx and hydrogel. Secondly, the natural composite provided a good microenvironment with the bottleneck effect of the silica sol-gel to prevent the enzyme from leaking [The synergistic action of PtNPs, MWNTs and sol-gel of CS-SiO leaking . Another leaking .3O4-Au) magnetic nanoparticles and GOx via cross-linking for blood glucose level estimation [3O4-Au nanoparticles facilitated the oxidation of H2O2 at the electrode and exhibited a good selectivity and fast amperometric response. This third generation of sensor technology, the GOx was directly immobilized onto the Gr electrode to improve shelf-life and reusability for practical application in clinical analysis. A potentiometric glucose sensor based on the CS-GOx-Fe3O4NPs composite modified gold coated glass electrode was reported by Khun et al., 2012 [Gold nanoparticles (AuNPs) decorated CS-GR nanocomposites film was prepared and characterized by Shan et al., 2010 . The goltimation . The CS-l., 2012 . This wol., 2012 . Howeverl., 2012 .Dip coating/dipping, spin coating, and blade coating methods are also used for creating various low-cost configurations at electrode surface. Self-assembly of PANI-grafted CS/GOx nanolayered films prepared by LBL-dip coat technique for electrochemical biosensor applications were reported by Xu et al., 2006 . These h2O2) were added for wider applications in development of pH-sensitive composite films for sensing. Authors have achieved simple, fast, uniform, and controllable co-deposition of CS-hydrogel, GOx, MWCNTs for constructing biosensors [Electrodeposition is another conventional surface modification method to improve the surface characteristics and functional properties of a wide variety of materials including biomolecules ,146,147.osensors . In anotosensors .2NPs) and entrapped GOx, this electrode showed high resistance to the ascorbic acid (AA) during electrochemical glucose estimation. CS-stabilized MnO2NPs directly suppressed the interfering signals of ascorbates by converting AA to an inactive product before it reached the electrode surface. Thus, the proposed CS-GOx/ MnO4NPs/AuDE sensor is another possibility for the fabrication of interference-free glucose biosensors [Gold disk electrode (AuDE) was modified by CS film integrated with manganese dioxide  to smooth the ruff electrode surface using CS matrix. In some cases, entrapment/encapsulation/electrochemical deposition/electropolymerization have been performed using hybrid solution of enzyme-chitosan and nanomaterial to construct mono or multi-layered film structure ,158,159.3+ side groups to form self-assembly for direct electron wiring to enhance the DET, as reported by Kumar-Krishnan et al. [3O4@SiO2 nanoparticles (Fc-AFSNPs) could be effectively employed for the fabrication of reagentless glucose sensors by forming CS-GOx-enzyme nanocomposite films on magnetic carbon paste electrodes (MCPEs) [2O2 but also inhibited the responses of interferents at a much lower applied potential [A SAMs-based glucose biosensor was fabricated in stepwise process where two layers were casted on the surface of gold electrode (AuE). The main problem during electrochemical studies is peeling-off of enzyme film located at the electrode surface due to weak non-covalent interactions between GOx and Au surface. Therefore, strong bonding or protective layer is required to form SAMs. When the CS at a pH above its pKa value (6.3) it will deprotonate the primary amino groups and become insoluble with retention of its natural properties i.e., film forming ability and biocompatibility ,158. Then et al. ,102. Then et al. . Silver  (MCPEs) . For thi2 group of chitosan while other –CHO group is covalently linked to –NH2 groups on the surface of GOx. This mulit-layered electrode modification afforded excellent microenvironment for rapid biocatalytic reaction to glucose in real sample [The alternate adsorption of polyanions and polycations with integration of different materials such as nanomaterials, proteins/ enzymes in matrix onto solid substrates in the form of thin film is so called the layer-by-layer (LBL). LBL is a self-assembly-driven surface modification strategy which allows the construction of multilayered nanostructured films onto substrates of any geometry, from simple bi-dimensional surfaces to more complex 3-D porous scaffolds . Multi-ll sample .Biosensor with sol-gel matrix mostly suffer from the limited diffusion, slow response time and restricted electron transfer. The sandwich configuration can be better option to overcome these limitations where enzyme layer is protected by top and bottom layer of composites or nanocomposites. The typical sandwich configuration at electrode allows homogenous distribution of enzyme and nanomaterials with controlled matrix porosity. High amount of active enzyme triggers fast enzymatic catalysis and layered porous matrix provides rapid diffusion of substrates and analytes required ,163. MiaIn some cases, a separate film/ membrane containing enzyme was placed on the surface of a moist dialysis membrane as a lamination layer and fastened with an O-ring . While i2. Artigues et al., proved that sensor architecture utilizing titanium dioxide nanotube arrays (TiO2NTAs) not only has features like simplicity and low cost but also can be used for measuring glucose in four different food products, including soft drinks, soy sauces, dairy products and tomato sauces [Conventional electrodes suffer from sensitivity and sluggish electron transportation. Electrode micro-fabrication technology offers great way to overcome these issues. Such microelectrodes (µ-electrode) deal with issues like diffusion controlled current, low charging current, high signal to background ratio (Faradaic/charging), and reduced solution-resistance due to their highly compact-arrayed design with bulk production of multi-electrodes ,166. Mulo sauces .2NTAs. GOx with CS-Neodymium orthophosphate nanoparticles (NdPO4NPs) composite with high specific surface area offered sensitive, selective and stable enzymatic biosensor for determination of glucose in human plasma. The CV and EIS studies revealed that the modified electrode can also be used to immobilize other redox enzymes due to composite configuration [This nanotube array-based amperometric glucose sensor was constructed by physical immobilization of GOx in CS hydrogel onto highly ordered TiOguration . The anaLike CS and CT precursors, their micro and nano-form are also promoting the compatibility with natural surroundings to the biological counterpart during conjugation ,157,172.Dilute acid hydrolysis of chitin at high temperature resulted into nano-whiskers (CTNWs) with slender parallelepiped rods ,183. TheNanochitosan has very low toxicity and excellent adhesion property for enzyme immobilization and interface design for various applications ,189,190.The reactive functional groups and surface charge of chitin have been successfully utilized for preparation of a wide spectrum of its derivatives such as chitosan, alkyl chitin, sulfated chitin, dibutyryl chitin carboxymethyl chitin, and nanostructured chitin/chitosan with high commercial values. However, there are limited studies based on chitin nanocomposites for biosensing application due to its insolubility in organic and inorganic solvent, low metal ion capacity, separation difficulty, non-tunable porous structure and low surface area. Still there is strong need for the development of simple and easy synthesis methodology for chitin composites and nanocomposites that can be used for applications for industrially important enzyme immobilization and practically useful biosensor fabrication. Ecofriendly and renewable chitinous materials when redesigned with nanomaterials, have the potential to become new, superior and advance materials. These nanocomposites could possess important properties such as compatibility, biodegradability, and mechanical strength for biosensors. In the future, these innovative interfaces with further tuning can be used for the modification of different kinds of transducers for versatile sensing with relatively short analysis time.


Adults with short stature have been previously reported to have increased risk of cardiovascular events and hyper-LDL-cholesterolemia. We aimed to assess the association between height and lipid profiles among Korean adolescents and adults. We analyzed data from the Korea National Health and Nutrition Examination Survey from 2007 to 2015, from 37,889 individuals (aged 12–59 years). In adolescents, total cholesterol (TC) and low density lipoprotein-cholesterol (LDL-C) levels had profound associations with height in both boys and girls, while high density lipoprotein-cholesterol (HDL-C) levels had an inverse association with height only in boys. Height was inversely associated with TC, triglycerides (TG), and LDL-C concentrations in men and women and positively correlated with HDL-C concentration in women. In boys, the odds ratios (ORs) for hypercholesterolemia, hypertriglyceridemia, hyper-LDL-cholesterolemia were higher for shorter subjects (ORs = 2.38~7.01), while only the OR of hyper-LDL-cholesterolemia was significantly higher in girls with short stature (OR = 3.12). In adults, the ORs for hypercholesterolemia, hypo-HDL-cholesterolemia, and hyper-LDL-cholesterolemia were significantly higher in short subjects than in tall subjects after controlling for covariates (ORs = 1.50~2.61). Also, short men showed significantly higher ORs for hypertriglyceridemia (OR = 1.85) than tall men. Short stature was significantly associated with adverse lipid profiles in both adolescents and adults. Another meta-analysis with 1 million subjects reported that the risk of fatal or non-fatal coronary artery disease (CAD) decreases by 8% per every 6.5 cm increase in height2. Similarly, previous studies from Korea have reported a negative association between height and all-cause mortality4.Height is recognized as an important indicator that reflects genetic and environmental factors, including nutrition, hormonal regulation, pubertal development, and chronic disease during the fetal-to-adolescent period. Following the first report in 1951 of the increased risk of coronary heart disease (CHD) with short stature in adults, this inverse association between adult height and cardiovascular disease (CVD) risk has been supported by the results of several prospective large-scale studies and meta-analyses. A meta-analysis of the relationship between height and CHD revealed that short adults had ~1.5 times higher risk for CHD morbidity and mortality than adults with tall stature7. Lipid levels in childhood often track to adulthood. Although CVD usually develops in later life, it is well known that the accumulation of intimal fatty streaks, which are early atherosclerotic lesions, develops from childhood in association with dyslipidemia. Furthermore, dyslipidemia often accompanies other CVD risk factors including insulin resistance and hypertension, which exacerbate atherosclerosis. The identification of risk factors for dyslipidemia in children and adolescents is therefore essential for its early detection and management and the consequent prevention of CVD in later life8. In Korea, the overall prevalence of dyslipidemia in adolescents aged 10–18 years is 21.7~25.2%; prevalence is reported to be two times higher (53.1~56.1%) in obese adolescents9. However, few studies have been conducted on the relationship between height and blood lipid concentrations in children and adolescents15. Specifically, there have been no studies on the association of dyslipidemia and height in Korean adolescents and adults. In the present study, we therefore aimed to assess the association between height and serum lipid profile in Korean adolescents and adults using data from the Korean National Health and Nutrition Examination Survey (KNHANES).Dyslipidemia is one of the major established risk factors for CVD; treatment of dyslipidemia ameliorates the risk of CVD, and dyslipidemia has been reported to be inversely associated with adult heightThe mean age of the adolescents was 15.1 years, and the mean age of adult men and women were 38.9 and 39.3 years, respectively , the OR in women with heights less than 10th percentile was 1.33 (0.86–2.04). In men, the OR for hypertriglyceridemia (OR = 1.85) was significantly increased in shorter subjects than in taller subjects in men, while such an increase in OR was not statistically significant in women (Table The odds ratios (ORs) and 95% confidence intervals for dyslipidemia according to the height percentile are presented in Tables The present study found that short stature has a significant inversely association with dyslipidemia in both Korean adolescents and adults. In adolescents, this inverse relationship between height and dyslipidemia showed sex-related differences. The risk of hypercholesterolemia, hypertriglyceridemia, and hyper-LDL-cholesterolemia was linked with height in boys, while only hyper-LDL-cholesterolemia was significantly related to height in girls. In adults, all four types of dyslipidemia were confirmed to be negatively affected by short stature in both men and women.10, a few prospective studies have analyzed the association between height gain and LDL-C levels in adolescents. The Bogalusa Heart Study reported that changes in height for five years in boys were inversely correlated with TC, LDL-C, and HDL-C levels12 (9). This negative correlation between the LDL-C concentration and growth rate in both boys and girls was also supported by the findings of large-scale prospective study conducted in Japan14. Finally, a recent study of 6,300 adolescents conducted by the West Virginia Coronary Artery Risk Detection in Appalachian Communities (CARDIAC) project reported that the risk of hyper-LDL-cholesterolemia increases significantly in the first (shortest) quartile stature compared with that in the fourth  quartile stature (OR = 1.3~3)15. Consistent with previous studies, the present study also found that shorter adolescents had higher TC and LDL-C levels, with significantly increased OR for hyper-LDL-cholesterolemia (OR = 3.12~4.35). Unlike those on LDL-C, studies on the association of TG with height are rare. A previous cross-sectional study in children and adolescents aged 13 to 18 years from the UK demonstrated no significant association between height and TG concentration10. Similarly, a longitudinal study among boys in the USA found no association between changes in TG levels and rapid height increase12. In contrast to this previous study, the present study found statistically higher OR for hypertriglyceridemia in shorter boys, and this association was not observed in girls. It is also important to note that obesity indices were significantly higher in boys with taller heights than in boys with shorter heights in this study population. These results suggest that the adverse effect of short stature on CVD risk in adolescents might be comparable or even stronger than obesity indices, especially for boys.Since a small cross-sectional study from the 1980s reported a negative correlation between height and TC in boys12. Another study from Japan reported a negative correlation between HDL-C levels and height in both sexes in the age of 10–14 years13. In the present study, height was negatively correlated with HDL-C levels, only in boys, but its association with hypo-HDL-cholesterolemia was not statistically significant after adjusting for relevant covariates. In contrast with adolescents, adults with shorter height percentile showed a significantly higher risk of hypo-HDL-cholesterolemia, which might be explained by the effects of aging on HDL metabolism. Increased insulin resistance and systemic inflammation, a decline of sex hormone, and impaired lecithin cholesterol acyltransferase activity due to cellular senescence16 may enforce the inverse relationship between height and HDL-cholesterol in adults. The results of studies on the relationship between height and dyslipidemia/lipid concentration in adults have been inconsistent. Two prospective cohort studies from Europe reported an association between greater leg length and favorable levels of TC, LDL-C, and HDL-C at 53 years in UK adults6 and between taller stature and lower non-fasting TC and non-HDL-C levels in middle-aged men5. These two studies did not include TG concentration. In a study of individuals aged 50 years and older in China, height was negatively correlated with both LDL-C and HDL-C levels and positively correlated with TG concentration7. In a hospital-based study of 3,016 patients aged 30–59 years in Japan, no significant correlation was found between height and individual lipid profiles, but the OR for overall dyslipidemia in non-obese male patients was significantly decreased in taller subjects17. The present findings suggest that height in adulthood is favorably associated with not only LDL-C and HDL-C levels, but also TG levels. This finding is consistent with that of a recent genome-wide association study, which reported that increased height-raising allele was associated with decreased LDL-C and TG levels18.Unlike the negative correlation between height and LDL-C in both boys and girls, which is reported by most studies, reports on the relationship between height and HDL-C are inconsistent. In previous studies from the UK and USA, HDL-C levels were negatively associated with height increase in boys, but not in girls, aged 8 to 18 years19. Unlike alcohol consumption with conflicting effects on CVD risk, beneficial effects of physical activity on the lipid profiles, including lowering LDL-cholesterol and triglycerides, and increasing HDL-cholesterol have been consistently reported by previous studies20. To eliminate the possible favorable impact of these lifestyle factors on lipid profiles, we included these confounding factors for final adjustment. Another thing that we have to note is an inverted J-shaped association between OR for hyper-HDL-cholesterolemia and height in women. We could not find any difference in age and confounding factors between the shortest women (height <10th percentile) and the next shortest women (height 10~29th percentile). There has been no report of an inverted J-shaped association between height and HDL-cholesterol similar to our result. In overall, a favorable trend for hypo-HDL-cholesterolemia according to the height increase was found in women. Therefore, further research is needed to determine whether the risk of hypo-HDL-cholesterolemia is rather alleviated in severe short stature compared with mild short stature in women.An interesting fact in this study population was that taller adults tend to have higher alcohol consumption and physical activity compared with shorter adults. Higher alcohol consumption had been related to higher HDL-cholesterol in previous studies, but its beneficial effect on CVD risk is known to be alleviated by TG-elevating effect of alcohol21. Also, estrogen promotes reverse cholesterol transport process, such as hepatic secretion of cholesterol into bile, and reduces hepatic TG content through hepatic estrogen receptor α21.There were gender-related differences in the relationships between height and lipid profiles. In both adults and adolescents, lipid profiles in males tended to be more clearly affected by short stature. Boys with short stature showed a remarkably increased risk of both hyper-LDL-cholesterolemia (OR = 4.35) and hypertriglyceridemia (OR = 2.38), while girls with short stature showed an increased risk of hyper-LDL-cholesterolemia only (OR = 3.12). Likewise, the unfavorable effects of short stature in hypertriglyceridemia and hypo-HDL-cholesterolemia were more evident in men than in women. This gender difference can be explained by the beneficial effects of estrogen on lipid metabolism. Estrogen increases the accumulation of subcutaneous fat and restrains the increase of visceral fat, consequently allows a metabolically favorable distribution of adipose tissue23. In addition, it is well known that thyroid hormone plays an important role in hepatic synthesis and metabolism of fatty acids and cholesterol, and hypothyroidism leads to hypercholesterolemia and hypertriglyceridemia24. Therefore, it can be inferred that adolescents with adequate GH and thyroid hormone secretion are likely to have optimal growth and lipid profiles. In addition, since cholesterol is an essential component of cell membranes and steroid hormones, serum cholesterol may be lowered in individuals with a high growth velocity due to increased cholesterol consumption12. The skeletal system is now recognized as an important endocrine organ, which is involved in lipid metabolism. Several studies have shown that blood fatty acids are taken up by the skeletal system and are used as an energy source by the osteoblasts, thereby clearing circulating lipoproteins and non-esterified fatty acids25. In addition, osteocalcin, an osteoblast-derived hormone, increases the expression of the adiponectin gene in adipocytes26. Adiponectin causes an increase in serum HDL via inducing an increase in the hepatic production of apolipoprotein A-1 and the ATP-binding cassette transporter A127. Additionally, adiponectin lowers serum TG by enhancing VLDL catabolism in skeletal muscle adipose tissue27. Therefore, high osteocalcin levels in adolescents with high growth rates28 may result in favorable lipid profiles29. Finally, as noted above, a recent genome-wide association study reported a correlation between genetically determined short height and increased risk of coronary artery disease, which is partly explained by an association between height-related single nucleotide polymorphisms and lipid profiles, especially those of LDL-C and TG18.The mechanisms supporting the link between height and dyslipidemia are not currently clear. Two hormones that play a decisive role in bone growth are growth hormone (GH) and thyroid hormone, both of which contribute to the improvement of lipid profiles. The lipolytic action of GH has been well described, particularly on visceral fat, and the administration of recombinant human GH in children and adults with GH deficiency or obesity has been reported to improve lipid metabolism by reducing TC and LDL-C levelsOur study does have some limitations. As this was a cross-sectional study, causality could not be inferred between short stature and dyslipidemia. In addition, we did not examine some confounding factors, such as pubertal stage, dietary intakes and family history of dyslipidemia. Nonetheless, to the best of our knowledge, this is the first study that reports the association of the profiles of all lipids, including TG, with height in adults as well as in adolescents, using nationally representative data. We tried to minimize the age-associated bias by using age or age-group specific height percentile. We also included various covariates including age, waist circumference, blood pressures, fasting glucose, and lifestyle factors, that are known to be potentially related to lipid profiles in the statistical analysis.In conclusion, this study presents the evidence of short stature as associated with adverse lipid profiles in both adolescents and adults, and also suggests possible mechanisms to explain this association. Our results may help partly explain the association between short height and increased risk of CVD.We analyzed data from the Korean National Health and Nutrition Examination Survey (KNHANES), conducted by the Korean Centers for Disease Control and Prevention between 2007 and 2015. This nationwide cross-sectional survey includes demographic and anthropometric information, lipid profiles and information on health-related behaviors, including alcohol intake, physical activity and household income. We limited the analyses to individuals aged 12–59 years. Among the potentially eligible 44,448 individuals, we excluded participants for whom incomplete data were available for lipid profiles and anthropometric measurements (n = 5463). Other exclusion criteria included serum triglycerides ≥400 mg/dL (n = 784); medical history of liver cirrhosis, nephrotic syndrome, or endocrine disorders; and use of medications affecting blood lipid concentrations (n = 312). Eventually, 37,889 individuals aged 12–59 years who completed blood tests for lipid profiles after an overnight fast were enrolled in this study, comprising 5,207 adolescents  and 32,682 adults . The KNHANES protocol was approved by an Institutional Review Board of the Korean Centers for Disease Control and Prevention , and conducted according to the Declaration of Helsinki. All participants or their parents/guardians provided written informed consent form. This study was approved by the institutional review board of Inje University, Sanggye Paik Hospital (IRB No. SGPAIK 2019-02-005), which waived the requirement for informed consent due to the anonymity of the data obtained from the KNHANES dataset.th rib and the superior border of the iliac crest. Systolic and diastolic blood pressure were measured using a standard mercury sphygmomanometer on the right arm on three occasions while the participants in a sitting position after they rested for more than 10 minutes. The final blood pressure values used for analysis were the average values of three measurements.Participant height and body weight were measured to the nearest 0.1 cm using a stadiometer and 0.1 kg using a digital weight scale, respectively. BMI was calculated as the weight (kg) divided by the square of the height (m). Waist circumference was measured following a full expiration at the narrowest point between the bottom of the 1030. In 2008–2015, serum fasting glucose and lipid profiles, including LDL-C, were determined enzymatically using a Hitachi Automatic Analyzer 7600 . Participants were categorized as five groups according to the height percentile . Sex and age-specific height percentile was applied for adolescents, and sex and age group-specific  height percentile was applied for adults. The sex and age-specific height percentile values were presented Supplementary Table Blood collections were performed via venipunctures from the antecubital fossa after a fasting period of at least 8 h. In 2007, serum concentrations of TC, HDL-C, TG and fasting glucose were measured enzymatically using an ADIVIA 1650 . Serum LDL-C levels were calculated using Friedewald’s formula31. In adults, hypercholesterolemia (≥240 mg/dL), hypertriglyceridemia (≥200 mg/dL), hyper-LDL-cholesterolemia (≥160 mg/dL), and hypo-HDL-cholesterolemia (<40 mg/dL) were determined according to the Korean Society of Lipid and Atherosclerosis guidelines for adults32.Dyslipidemia was defined as the presence of at least one of the following: hypercholesterolemia, hypertriglyceridemia, hyper-LDL-cholesterolemia, and hypo-HDL-cholesterolemia. In children and adolescents, hypercholesterolemia (≥200 mg/dL), hypertriglyceridemia (≥130 mg/dL); hyper-LDL-cholesterolemia (≥130 mg/dL), and hypo-HDL-cholesterolemia (<40 mg/dL) were defined per the National Heart, Lung, and Blood Institute’s (NHLBI) Expert Panel GuidelinesStatistical analyses were performed using SPSS version 21.0 . The complex samples descriptive procedure was used to evaluate the mean and standard error of scale variables. The complex samples crosstabs procedure was used for categorical or ordinal variables. The complex samples general linear model was used to compare serum concentrations of lipid profiles according to the height percentile and to calculate P values for trend. Multiple logistic regression analyses were used to calculate the ORs for dyslipidemia according to the height percentile after adjusting for covariates including age, waist circumference, systolic/diastolic blood pressure, fasting glucose, household income , physical activity , alcohol consumption , and menopausal status . TG levels were log-transformed owing to their skewed distribution. For all analyses, P-values were two-tailed and P < 0.05 was considered statistically significant.Supplementary Information


In randomised clinical trials, the type II anti-CD20 antibody obinutuzumab has been shown to be more effective than rituximab for therapy of chronic lymphocytic leukemia (CLL) and follicular lymphoma (FL). However, this enhanced efficacy was linked with elevated rates of high-grade adverse events. The aim of this study was to assess the tolerability and toxicity profile of obinutuzumab treatment in routine patients with CLL and FL, of whom the majority had experienced toxicity or resistance to rituximab.This retrospective cohort study investigated fifteen obinutuzumab-treated patients, eight with CLL and seven with FL. The course of the disease, comorbidities and treatment-related toxicities were recorded. All patients with CLL and all but three FL patients had any form of pre-treatment with rituximab.Between October 2014 and August 2017, 15 patients were treated with obinutuzumab at the University Hospital Krems. In the CLL-cohort, 1 patient  developed pneumonia, 2 (25%) febrile neutropenia, 6 (75%) anemia and 7  thrombocytopenia, respectively. One patient exhibited an infusion-related allergic reaction. In the FL-cohort, 6 patients  presented with thrombocytopenia, 3  with anemia and one patient with neutropenia. No sepsis or consecutive solid tumors were seen in any of the patients.Obinutuzumab was mostly well tolerated in mild to heavily pre-treated patients with CLL and therapy-naïve or pre-treated patients with FL. The frequency and profile of adverse events and toxicity was comparable to data from previous clinical studies and could be managed adequately in the setting of a University Clinic. The WHO-classification  describes chronic lymphocytic leukemia (CLL) as an indolent lymphocytic lymphoma, characterized by leukemic progression . It is awww.nccn.org) suggest that CLL does not require treatment until symptoms are present or disease progression, causing severe cytopenia, can be seen [The clinical staging systems devised by Rai et al.  and Bine be seen , 7. In c be seen . Patient be seen . If ther be seen –14.If del(17p) or TP53-mutations are present, immuno-chemotherapies show significantly less response-rates and therefore targeted therapies such as ibrutinib  –15, or iFollicular lymphoma (FL) is an indolent Non-Hodgkin lymphoma (NHL), characterized by a slowly progressive lymphadenopathy . FL is tThe clinical staging is performed by means of the Ann Arbor classification . Various®) and Prednisone-containing regimen) or bendamustine should be applied [The combination of rituximab and chemotherapy resulted in improved overall response rates (ORR), longer progression free survival (PFS) and OS of FL patients compared to treatment with chemotherapy alone . In orde applied . Rituxim applied  as a sho applied .®, MabThera®) is an indispensable cornerstone in treatment algorithms of B-cell malignancies. When combined with chemotherapy, rituximab could significantly improve ORR, PFS and, in some cases, OS of patients with FL, DLBCL, marginal zone lymphoma (MZL), mantle cell lymphoma (MCL), as well as B-CLL [The chimeric, type I anti-CD20 monoclonal antibody (mAb) rituximab , antibody-dependent cell-mediated cytotoxicity (ADCC), induction of apoptosis and sensitization to chemotherapy . Main ad® in USA and Gazyvaro® in Europe) is a novel, type II, glyco-engineered, humanized anti-CD20 monoclonal antibody. It has been developed to enhance efficacy in patients with B-cell malignancies by inducing higher levels of immune cell-mediated tumor cell killing and direct cell death when compared with rituximab [Obinutuzumab  or FL (n = 7) were treated with obinutuzumab-bendamustine and followed up at the University Hospital Krems. In our CLL patient cohort, gender distribution was slightly shifted towards male sex, while in the FL subgroup female sex was more frequent. The median age at start of obinutuzumab therapy was 68: in detail 71 years for patients with CLL and 65 years for patients with FL, which is very similar to other published patient cohorts [This retrospective, single-center study analyzed the tolerability and toxicity of obinutuzumab in patients with CLL or FL. Between October 29 cohorts , 42. We Previous randomized clinical trials evaluating the efficacy and safety of obinutuzumab in NHL were performed in both untreated  and pre-Preterm termination of rituximab therapy might be necessary in some cases. Four out of eight (50%) CLL patients of our cohort had terminated rituximab therapy early because two of them showed an infusion-related allergic reaction. Of note, this small group of patients tolerated obinutuzumab without evidence of an infusion-related allergic event and both completed the treatment regimen as prescribed. Of the remaining two patients who had stopped rituximab, one female patient developed thrombocytopenia, the other  patient experienced anemia as well as thrombocytopenia while obinutuzumab was applied. Both, thrombocytopenia and anemia were also commonly reported in previous clinical trials , 42. InfIn the FL group, four out of seven  patients had received rituximab. One of them had stopped rituximab therapy early because of an allergic reaction but tolerated obinutuzumab well. However, this patient developed anemia and thrombocytopenia during obinutuzumab therapy. Sehn et al. found that obinutuzumab plus bendamustine followed by obinutuzumab maintenance therapy in rituximab-refractory patients with indolent NHL resulted in serious adverse events in 38% and death due to adverse events in 6% of the patients, respectively. Neutropenia (33%), thrombocytopenia (11%), anaemia (8%) and infusion-related reactions (11%) were observed in this trial . TherefoNone of our patients underwent stem cell transplantation, which is partly due to the fact, that stem cell transplantations are not performed at the University Hospital Krems, and thus patients are referred to and treated at other hospitals. However, one male patient was offered stem cell transplantation, but refused out of personal reasons. Two out of 15 patients died during or after completing the therapy with obinutuzumab. These deaths were not therapy-related, as one had a second haematological malignancy, a Multiple Myeloma, which was unveiled during the obinutuzumab-bendamustine therapy. The other patient developed after an initially good response an early relapse with a CD20-negative CLL-clone.Our findings are in agreement with large controlled trials showing that obinutuzumab is effective in both patients with CLL or FL but without significant impairment of healthy-related quality of life indices . As thisIn conclusion, obinutuzumab was mostly well-tolerated in a group of mild to heavily pre-treated patients with CLL and therapy-naïve or pre-treated patients with FL. Therefore, the planned obinutuzumab-bendamustine regimen could be completed in the majority of patients offering an effective therapeutic alternative to patients refractory to standard therapies. The frequency and profile of adverse events and toxicity was comparable to data from previous clinical studies and could be managed adequately in the clinical setting of a University Clinic. Nonetheless, further studies are needed to characterize the risk of severe adverse events due to obinutuzumab therapy and their appropriate management.Age (at the start of obinutuzumab-bendamustine therapy).Gender.Demographic data.Stage of the disease.Risk score.Progression-free survival (PFS).Pre-treatment (type and quantity of pre-treatments).Stem cell-transplantation.Comorbidities.Patient characteristics.Infectious diseases, pneumonia, sepsis, tumour lysis syndrome, allergic reactions.Need for erythrocyte transfusion, thrombocyte transfusion.Laboratory parameters .Leukopenia, neutropenia, febrile neutropenia, anaemia, thrombocytopenia.Patient's adverse effects (staged according to the to the Common Terminology Criteria for Adverse Events Version 5.0 (CTCAE v5.0) of the National Cancer Institute (NCI) of the USA):Disease-specific parameters.The design of this study is an uncontrolled retrospective cohort study using data from the database of the University Hospital Krems: the study-participants were pseudonymised and got a consecutive serial number (001 - 002 - …). The analysis of the study-oriented data took place only with those assigned serial numbers. The following parameters were recorded and used for statistical evaluation:The registry was approved by the ethics committee of the Karl Landsteiner Private University in June 2017.Participants of the study were patients with CLL (n = 8) and FL (n = 7), who were treated with obinutuzumab-bendamustine after a rituximab-containing therapy in the period of 2014 to 2017 at the Department of Internal Medicine 2 of the University Hospital Krems. The sample size of the study was 15 participants. Exclusion criterion was age below 18 years.2 body surface area at days 1 and 2. During the first cycle, obinutuzumab was administered in two doses: 100 mg at day 1, and 900 mg at day 2 plus 1000 mg at days 8 and 15.All patients received immuno-chemotherapy with obinutuzumab and bendamustine for 6 cycles at intervals of 28 days. During each cycle, patients received 1000 mg obinutuzumab at day 1 and 90 mg bendamustine /mp<0,05.The collected data was descriptively analysed using Microsoft Excel (Version 16.9), Microsoft Word (Version 16.9.1) and GraphPad Prism (Version 7.0d). Initially, descriptive analyses of gender and age were performed. Subsequently, laboratory values before and after obinutuzumab therapy were collected and median as well as minimum and maximum values were analysed. Scatter dot plots were chosen to demonstrate the range between minimum and maximum values of leukocyte counts (WBC), haemoglobin values (Hb), thrombocyte counts (PLT) and lactate dehydrogenase levels (LDH). Laboratory values after therapy were determined at the first control visit at our outpatient clinic after having completed immuno-chemotherapy with obinutuzumab-bendamustine. This means that these values are in the FL group before the start of obinutuzumab maintenance therapy. Respective laboratory values before and after therapy were analysed by means of paired t-test, and statistical significance was accepted at This work is based on the bachelor's thesis of Theo Pirich, submitted in May 2018 at the Karl Landsteiner University of Health Sciences entitled “Tolerability of obinutuzumab therapy in patients with rituximab-relapsed/refractory B-cell malignancies - a retrospective uncontrolled cohort study”.