Intravenous fluid administration is an essential component of sepsis management, but a positive fluid balance has been associated with worse prognosis. We analyzed whether a positive fluid balance and its persistence over time was an independent prognostic factor in septic patients.We prospectively studied fluid intake and output for 7 days in 173 consecutive adult patients treated for sepsis in our Department of Intensive Care.p = 0.03), but output volumes were similar. As a result, the daily fluid balance was more than twice as large in the non-survivors as in the survivors . Persistence of a positive fluid balance over time was associated with increased mortality. Using a multivariable time-dependent Cox model, a positive fluid balance was independently associated with higher mortality .Of the 173 patients, 59 died (34 %). Mean daily fluid intake was higher in non-survivors than in survivors  contains supplementary material, which is available to authorized users. Sepsis, considered today as a dysregulated inflammatory response to an infection , is respThis prospective observational study was performed in the 35-bed Department of Intensive Care at Erasme University Hospital, Brussels. The study was approved by the ethics committee of Erasme Hospital (reference P2013/108), who waived the need for informed consent due to the observational nature of the study.All adult patients admitted to the department during 2012 were included if they met the following criteria: (a) age older than 15 years; (b) suspected or proven infection supported by clinical evidence and/or positive bacteriological data, and treated with antibiotics; (c) sepsis-associated organ failure, as defined by a Sequential Organ Failure Assessment (SOFA) subscore of 3 or 4 ; (d) durPatients were treated according to department policy using the Surviving Sepsis Campaign guidelines . Fluid aDemographic and bacteriologic data were collected from all patients, as were all relevant elements needed to calculate the SOFA score. We also noted the duration of hospital stay before ICU admission, medical or surgical (emergency or elective) reason for admission, origin , length of ICU stay, ICU and hospital survival. The use of diuretics or renal replacement therapy  was also noted.Daily fluid intake was calculated as the sum of all intravenous and oral fluids. The daily fluid output was calculated as the sum of the volumes of urine output, ultrafiltration fluid, drain fluid, and estimated gastrointestinal losses (including stools only in the presence of profound diarrhea). Insensitive losses were not taken into account because they are difficult to assess reliably. Daily fluid balance (according to baseline patient weight) was calculated by subtracting the total fluid output from the total intake. Day 1 was defined as the time between ICU admission and the next morning.t test, Mann-Whitney test, chi-square test or Fisher’s exact test, as appropriate. Repeated measurements were compared using linear mixed models. Time-dependent Cox models were performed to assess the effect of daily fluid balance on survival in the whole population and in the subgroup of patients with septic shock. Daily fluid balance measurements were considered as the time-dependent covariate. The main effect model was built using a backward stepwise elimination technique. The variables considered in the multivariable modeling were selected based on their p value in univariate analysis. The threshold considered was 0.1 as a compromise between the number of variables that could be considered in the multivariable analysis and the number of death events in the analyzed population. Colinearity between variables was checked before modeling. The results are presented as crude and adjusted hazard ratios  with 95 % confidence intervals (95 % CI). Statistical analyses were performed using IBM SPSS 20 statistical program for Windows . All statistical tests were two-sided and p values of less than 0.05 were considered statistically significant.Data are given as means with standard deviation or medians and interquartile ranges for continuous variables and as numbers and percentages for categorical variables. The Shapiro-Wilk test was used, and histograms and normal–quantile plots were examined to verify whether there were significant deviations from the normality assumption of continuous variables. Difference testing between groups was performed using Student’s During the study period, 225 patients were treated for sepsis in our department, of whom 52 were excluded because they had an ICU stay <48 hours. Accordingly, we studied 173 patients, 114 (66 %) of whom survived the ICU stay , the non-survivors had a longer shock duration  than the survivors.As expected, the non-survivors had a higher SOFA score on admission than the survivors, and were more likely to have a comorbid cancer or an infection involving Aspergillus Table . In patip = 0.03), but the difference in fluid output was not significant (p = 0.49). Overall, the daily fluid balance was more positive in non-survivors than in survivors (p <0.01). In the first hours of treatment, the fluid balance was similar in survivors and non-survivors, but from the second day was more positive in non-survivors than in survivors .In the survivors, the mean fluid balance became negative between days 4 and 5 and remained negative Fig. . The flup = 0.038), the fifth day  and the sixth day  , while RRT was used in 23 (39 %) non-survivors and 18 (16 %) survivors (p = 0.001). We separated patients into those who did not receive diuretics or RRT (“spontaneous diuresis”), those who received diuretics and those who received RRT. In all three groups, non-survivors had a higher daily fluid balance than survivors (Table Diuretics were administered in 24 (41 %) non-survivors and 33 (29 %) survivors (p <0.001), a finding which persisted after multivariable modeling . Other variables that were significantly associated with ICU mortality were age, the type of admission and the presence of cancer. In the subgroup of patients with septic shock, a positive fluid balance was also associated with ICU mortality .Using a time-dependent Cox model in which the daily fluid balance was considered as the time-dependent covariate (see Additional file We studied a single-center, medical-surgical population of 173 septic patients, 78 % of whom had septic shock, with an overall ICU mortality of 34 %. A positive fluid balance was independently associated with an increase in the risk of death. We also observed a relationship between the change in fluid balance over time and mortality.A positive association between fluid balance and mortality is quite well established. Results from the SOAP study, an observational study of 3,147 adult patients from 198 European ICUs, indicated that, in patients with sepsis, fluid balance was an independent risk factor for mortality . Alsous However, it is important to consider a time-related relationship, because fluid administration is dynamic, changing according to the patient’s evolution. Recently, it has been suggested that fluid administration for patients in shock should be considered according to the ROSD mnemonic: rescue, optimization, stabilization, and de-escalation phases . We did In our study population, the fluid balance was initially quite similar in the survivors and non-survivors but the non-survivors received more fluids so that already from the second day, the fluid balance was more positive in the non-survivors. After initial resuscitation, less fluid was administered in both groups, and the fluid balance decreased steadily in the survivors but not in the non-survivors. The differences in fluid balance were due to a greater fluid input in the non-survivors rather than to a lower fluid output. Survivors were more likely than non-survivors to have a negative fluid balance early in their ICU stay, and a positive fluid balance was an independent prognostic factor for ICU mortality. The relationship between positive fluid balance and mortality was present regardless of whether or not diuretics or RRT were used.The single-center nature of our study may be seen as a limitation, but it can also be a strength by limiting variability in patient management as different centers may have different protocols for fluid administration and use of diuretics and RRT. Single-center studies may, therefore, have increased intrinsic validity.In critically ill patients with sepsis, a persistent positive fluid balance is quite strongly associated with an increased risk of death. This observation supports the suggestion that fluid administration needs to be carefully titrated after hemodynamic stabilization . FurtherIntravenous fluid administration is an essential component of sepsis managementAccurately determining ongoing fluid requirements in patient with sepsis can be difficultPersistence of a positive daily fluid balance over time is quite strongly associated with a higher mortality rate in septic patients


The primary textures of the SF1 halite typified by cumulate and chevron halite suggest halite deposited in a shallow saline water and halite Th can serve as an temperature proxy. In total, one-hundred-twenty-one Th data from primary and single-phase aqueous fluid inclusions with different depths were acquired by the cooling nucleation method. The results show that all Th range from 17.7°C to 50.7°C,with the maximum homogenization temperatures (ThMAX) of 50.5°C at the depth of 3028.04 m and 50.7°C at 3188.61 m, respectively. Both the ThMAX presented here are significantly higher than the highest temperature recorded in this region since 1954and agree with global temperature models for the year 2100 predicted by the Intergovernmental Panel on Climate Change.Climate changes within Cenozoic extreme climate events such as the Paleocene–Eocene Thermal Maximum and the First Oligocene Glacial provide good opportunities to estimate the global climate trends in our present and future life. However, quantitative paleotemperatures data for Cenozoic climatic reconstruction are still lacking, hindering a better understanding of the past and future climate conditions. In this contribution, quantitative paleotemperatures were determined by fluid inclusion homogenization temperature (T The trigger mechanisms and evolutionary processes of climate change always have been a focus for geoscientists. The Mesozoic and Cenozoic eras are considered to be one of the most important turning points of climate change when Earth's systems transitioned from the middle Cretaceous–early Paleogene typical “green house” conditions to “ice house” conditions after the middle–late PaleogeneThe continental and oceanic configuration during the middle–late Eocene was very similar to modern conditions13The Bohai Bay Basin is one of the most important oil and gas basins in China and hosted thick evaporite depositions during the Paleogene. Previous paleotemperature studies in this region mainly focused on palynology and indicated climate conditions changed from dry-hot to warm-humid from the fourth to first members of the Shahejie Formation (Eocene to Oligocene) as suggested by Li et al.17h of fluid inclusions in halite is closely related to the water or air temperature during deposition. This paper quantitatively determines paleoclimate characteristics by analyzing the Th of primary, single-phase aqueous fluid inclusions from halite in SF1 (probably late Eocene to early Oligocene). We used the cooling nucleation method to directly measure deposition temperatures of halite. This method has been proven to be very effective and has been widely used in reconstructing ancient climate from different geologic time periods, including Precambrian20212224hMAX) of halite fluid inclusions can represent the highest temperature of the water body in which the halite deposited and faithfully reflect the ancient climate. Thus, we chose ThMAX from all samples in SF1 as a record of paleotemperatures in the Shulu Sag during this time.The TThe Shulu Sag is located on the southern edge of the Jizhong Depression of the Bohai Bay Basin (North China), and surrounded by the Hengshui and Xinhe Faults on its northern and eastern margins, and the Ningjin and Xiaoniuchun uplifts along its western and southern margins . It is a26The Shahejie Formation can be divided into four members  from bottom to top, based on petrologic characteristics . The SF1h values can be good climate proxies.The SF1 discussed in this study is characterized by evaporite sequences consisting of thin clastic layers shaped like bamboo kont  and argillaceous limestone . Halite h of fluid inclusions in these crystals is analogous to ancient air temperatures11212223252021h of the two types of fluid inclusions can be used to study ancient air temperatures1119202122h of fluid inclusions can be used to interpret paleoenvironmental conditions.Primary fluid inclusions in SF 1 halite occur in chevron- or cumulate-type crystals . Chevronh analysis due to primary fluid inclusion in most of the halite pieces are few or absent. The Size of primary fluid inclusions  range from 2 to 20 μm and up to 40 to 50 μm. Primary fluid inclusions coexist in both single liquid and gas-liquid phases, and only the single-phase liquid fluid inclusions are chose for Th analysis. The Th of halite fluid inclusion was tested with the cooling nucleation method, and the results are shown in In total, about 360 pieces of halite from samples B493 and B1003 have been observed in detail and only 38 pieces of halite are available to ThAVG) of 39.8°C. Thirteen Th values were obtained from fluid inclusions in chevron crystals with ThAVG of 39.9°C, ThMIN of 24.4°C, and ThMAX of 50.5°C. Forty-eight Th values were obtained from fluid inclusions in cumulate crystals, with ThAVG of 39.7°C, ThMIN of 29.5°C, and ThMAX of 49.6°C. Sample B1003: Sixty-one Th values were obtained  higher than the air temperature3313hMAX values of these two samples  are very similar, we speculate that the higher temperatures state evidenced by Th of halite fluid inclusion in the Shulu Sag may last a considerable time. Combining with the Eocene paleotemperature data from other places in China11As mentioned earlier, the Th of fluid inclusions, halite samples were chosen by XRD . Temperature information is susceptible to distortion due to alteration of halite fluid inclusions by dissolution or heating during sawing. We referred to the methods outlined in Roberts and Spencerh using a Linkam THMSG600 heating and cooling stage. The heating rate of the stage was first set at 0.5°C/min, but was lowered to 0.1–0.2°C/min when approaching 20°C.Halite samples were collected from two depths (Y.J.Z., H.Z., C.L.L. and L.C.M. designed the research in the manuscript. B.K.L. and L.C.W. have been an active participant in the sample collection and processing. Y.J.Z. and H.Z. wrote the manuscript and prepared all figures. All authors reviewed the manuscript.


In the Funding section, the name of the “Natural Sciences and Engineering Research Council of Canada Discover Grant” is listed incorrectly. The correct funder name is: Natural Sciences and Engineering Research Council of Canada Discovery Grant.


In the Funding section, the project grant number from the funder Fundação para a Ciência e a Tecnologia (FCT) is missing. The correct grant number is: PTDC/AGR-GPL/100509/2008.


Since the invention of the atomic force microscope (AFM) three decades ago, there have been numerous advances in its measurement capabilities. Curiously, throughout these developments, the fundamental nature of the force-sensing probe—the key actuating element—has remained largely unchanged. It is produced by long-established microfabrication etching strategies and typically composed of silicon-based materials. Here, we report a new class of photopolymerizable hydrogel nano-probes that are produced by bottom-up fabrication with compressible replica moulding. The hydrogel probes demonstrate excellent capabilities for AFM imaging and force measurement applications while enabling programmable, multifunctional capabilities based on compositionally adjustable mechanical properties and facile encapsulation of various nanomaterials. Taken together, the simple, fast and affordable manufacturing route and multifunctional capabilities of hydrogel AFM nano-probes highlight the potential of soft matter mechanical transducers in nanotechnology applications. The fabrication scheme can also be readily utilized to prepare hydrogel cantilevers, including in parallel arrays, for nanomechanical sensor devices. Atomic force microscopy typically employs hard tips to map the surface topology of a sample, with sub-nanometre resolution. Here, the authors instead develop softer hydrogel probes, which show potential for multifunctional measurement capabilities beyond that of conventional systems. A compelling motivation in surface science is the development of nanoscale probes to seamlessly interrogate the molecular-level properties of material interfaces. This goal envisions probes which both engage and respond to the local material environment. To this end, scanning probe microscopy1With a growing range of AFM nanotechnology applications1114161719Realizing the potential of hydrogel composites as nanoscale actuating probes for AFM applications522242625Towards this goal, we explore the design, fabrication and testing of multifunctional hydrogel AFM probes. From a fabrication perspective, the design of hydrogel AFM probes represents a particularly challenging feature because it involves manufacturing and integrating hydrogel cantilevers and tips—the nanoscale geometrical features of the latter are also important. The learnings from work in this direction can be directly applied to fabricating tipless cantilevers as well. Experimentally, our findings indicate that hydrogel AFM probes enable stable and reproducible force, and imaging measurements of various inorganic and biological samples in air and liquid conditions. The combination of fabrication possibilities and multifunctional versatility afforded by the hydrogel probes offers the first demonstration of how 3D materials design strategies can be utilized for AFM applications, thereby showing the potential of soft matter to provide superior and versatile actuators for surface metrology. The hydrogel nano-probes developed in this work have the potential to be extended to cantilever-based nanomechanical sensing applications in both single measurement and array configurations.Hydrogel materials are readily fabricated by a molecular self-assembly process that involves light-sensitive polymerization reactions−1) was filled into the mould and the solution was cured by applying ultraviolet light  to compare contact mode AFM imaging performance, as described below. The mechanical force-sensing capabilities of the hydrogel cantilevers were determined by force-displacement measurements−1 which is a wider tuning range than that offered by commercial silicon-based cantilevers  at an identical contact force of 3 nN, and demonstrated that hydrogel and silicon probes had similar performances up to 5 Hz. However, at a 10-Hz rate, only the hydrogel probe maintained consistent image quality while significant distortions are observed in the image obtained by the silicon probe specifically designed for high-speed imaging in liquids , with phenylbis phosphine oxide (Sigma Aldrich) as the photoinitiator. The PEG-DA:photoinitiator weight ratio was 98:2. In most experiments, only one type of PEG-DA monomer was used with the average MW varied between 250 and 700 g mol−2; MA6, Karl Suss) for 15 and 10 s for 2015 and 2005 types, respectively. A commercial silicon cantilever handle  was used to define the cantilever handle mould. A PDMS pre-polymer mixture  was poured onto the two master moulds and cured on a hot plate at 100 °C for 60 min. The replica from the top cantilever mould was cut to have an inlet for injecting PEG-DA pre-polymer solution and aligned on top of the other replica from the bottom handle mould. The aqueous pre-polymer solution (98:2 weight ratio of PEG-DA and photoinitiator) was pipetted near the inlet and introduced into the aligned replica mould assembly via capillary action and cured by using a ultraviolet light-emitting diode source  for 5 s (ultraviolet dose: 590 mJ cm−2). After the top cantilever replica mould was removed, the cured PEG-DA cantilever was cut to the desired lengthReplica moudling was employed to fabricate hydrogel cantilevers . First, 3:HF:CH3COOH=95:2:3 v v−1 v−1), at room temperature for 5 min resulting in negative hemispheres. Then, the oxide layer was removed in 49% hydrofluoric acid and the wafer was cleaned with piranha solution, a mixture of sulfuric acid and hydrogen peroxide  and deionized water. 20 ml of the photoresist  mixed with 1 μl of a polystyrene microsphere suspension  was spin-coated  onto a 4-inch silicon wafer, and then baked on a hot plate at 120 °C for 1 min to embed the microspheres into a thin layer of photoresist. The wafer with negative pyramids or hemispheres and the wafer with embedded microspheres was silanized under vacuum conditions (−80 kPa) for 30 min by 95% trichlorosilane  before PDMS replica moudling. PDMS pre-polymer was poured onto silanized wafers and cured on a hot plate at 95 °C for 40 min. The cured PDMS substrate was peeled off and cut for preparing embedded sphere moulds  to leave exposed circular regions (10–20 μm in diameter). This exposed silicon region on the oxide-patterned wafer was etched anisotropically in 30 wt% aqueous potassium hydroxide solution at 70 °C for 15 min to form negative pyramids or etched isotropically in a mixture of hydrofluoric, nitric and acetic acids , human fibroblast cells  and HeLa cells  were used in experiments. Cells were maintained in Eagle's Minimum Essential Medium  supplemented with 10% foetal bovine serum  and 1% penicillin/streptomycin  at 37 °C in a humidified atmosphere containing 5% CO−1 was obtained. Considering the tip radius of a worn hydrogel nano-probe and the average etch rate, the processing time was determined for each hydrogel nano-probe.Worn hydrogel nano-probes with various tip radii were oxygen plasma-ashed by reactive ion etching  with a forward power of 43 W, oxygen flow rate of 6 s.c.c.m., and chamber pressure of 30 mTorr. With these processing parameters, an average etch rate of ∼10 nm minHow to cite this article: Lee, J. S. et al. Multifunctional hydrogel nano-probes for atomic force microscopy. Nat. Commun. 7:11566 doi: 10.1038/ncomms11566 (2016).Supplementary Figures 1-49, Supplementary Tables 1-5, Supplementary Discussion, Supplementary Methods and Supplementary References


The current state of the art for measuring stromal response to targeted therapy requires burdensome and rate limiting quantitative histology. Transcriptome measures are increasingly affordable and provide an opportunity for developing a stromal versus cancer ratio in xenograft models. In these models, human cancer cells are transplanted into mouse host tissues (stroma) and together coevolve into a tumour microenvironment. However, profiling the mouse or human component separately remains problematic. Indeed, laser capture microdissection is labour intensive. Moreover, gene expression using commercial microarrays introduces significant and underreported cross-species hybridization errors that are commonly overlooked by biologists.H&M array, and performed cross-species and species-specific hybridization measurements. We validated a new methodology for establishing the stroma vs cancer ratio using transcriptomic data.We developed a customized dual-species array, -16 for both, Mann-Whitney test). We confirmed the capability of the H&M array to determine the stromal to cancer cells ratio based on the estimation of cellularity index of mouse/human mRNA content in vitro. This new metrics enable to investigate more efficiently the stroma-cancer cell interactions (e.g. cellularity) bypassing labour intensive requirement and biases of laser capture microdissection.In the biological validation of the H&M array, cross-species hybridization of human and mouse probes was significantly reduced (4.5 and 9.4 fold reduction, respectively; p < 2x10These results provide the initial evidence of improved and cost-efficient analytics for the investigation of cancer cell microenvironment, using species-specificity arrays specifically designed for xenografts models. In spite of developments in high throughput molecular assessment biotechnologies, it remains particularly challenging to investigate molecular expression in the tumor microenvironment containing local tumor stoma, reactive stromal cells and cancer cells. There is increasing evidence that the development and progression of cancer is significantly affected by interactions between the tumor and its microenvironment -6, suggeLCM) [in silico estimates of cancer cells versus stromal cell ratios, which otherwise require labour intensive quantitative histology measures across hundreds of microscopic fields per tissue sample.Currently, the best way to selectively isolate cancer cells from a heterogeneous population of cells in a tumor is Laser Capture Microdissection (LCM) . While LLCM) -14, the LCM) . FurtherCross-Species Hybridization (CSH), which we have biologically confirmed to exist between human array probes with universal mouse RNAs [geneset-based approach is not designed to impute differential expression of individual mouse stromal genes. There is thus a need for improved species specificity of probes in newer arrays designed specifically for jointly studying stromal and cancer cell gene expression in xenograft models.Xenograft models, where human cancer cells are grown in immunodeficient mice, are popular for studying the tumor microenvironment. In such models, the genes of the stromal compartment and cancer cells come from distinct species: mouse and human respectively. While gene expression profiles of human cancer and mouse stroma have been analysed by human microarrays , this apuse RNAs . We alsouse RNAs . We prevuse RNAs . While mIn many tumor biology and pharmacology xenograft studies, the interplay and co-expression patterns of cancer and stromal cell genes need to be studied using the simultaneous analysis of both species in the same array experiment. When tissue expression is detected using separate mouse and human arrays, comparing mouse and human gene expression requires complex and artefact-inducing normalization of the two datasets. Currently, there is no genome-wide dual-species microarray available. While whole transcriptome RNA-sequencing (RNA-seq) of xenograft models provides an elegant alternative, the problem of disambiguating the expression intensities of homologuous genes remains (though the solution is quite different: an ambiguous RNA assembly problem). There exist two published dual-species partial arrays, in which one contains a mere 516 human probe-sets and 456 mouse probe-sets . Previouin vitro experiments . We used nine genomic datasets to compute human-mouse CSH . The list of housekeeping mouse genes was previously published [ftp://ftp.ncbi.nih.gov/pub/HomoloGene) on Feb, 2009 (Build 63). By design, these experiments generated Datasets S1, S2 and S3 that we provide for reuse by other groups at http://lussierlab.org/publications/HsMm_arrayMicroarray data from the two ts Table  are avaiublished ,23 and tDatasets S2, S3), which are redesigned to correct for comprehensive CSH. We designed two custom arrays: the second and final one improved on the original design after biological validation. The H&M arrays contain all legacy probes from the commercial human and mouse arrays as well as the newly designed ones . The second and final custom H&M array is identified as "H&M array version 2" . In an initial in vitro biological experiment, we cross-hybridize H&M array version 1 with mouse mRNAs, human mRNAs, and a combination of both human and mouse RNAs . Gene Ontology enrichment studies are also performed and show rectification of the cross-hybridization and validation of cellularity ratios .As shown in Figure s Figure , panel AAs shown in Table We first obtain human and mouse universal reference RNAs from ArrayIt , which were derived from a homogenate of all tissues from the organism, making them somewhat representative of any cell type. This universal RNA is amplified and labelled according to the Agilent Low RNA input Fluorescent Linear Amplification Kit protocol and according to Agilent's two-color quick amp labelling protocol. The two arrays are scanned with 5 µm resolution and 100% power gain at 600PMT for both Cy3 & Cy5 using the GenePix 4000B Axon Scanner.We quantify gene expression values using Agilent Feature Extraction Software and subtract the adjusted background for each probe from the mean expression value. Finally, we use an inclusion criterion to identify the probes that show some RNA binding where the minimum expression intensity has to be larger than a value of "1" when exposed to the pooled human and mouse RNA in both arrays. This threshold was set because when the expressed intensity was less than 1, probes vary widely across the two experimental repeats using the same quantity of pooled human and mouse RNAs.length of the alignments and (ii) the number of mismatches .The Basic Local Alignment Search Tool (BLAST) algorithm  is run wGS) and the parameters used in the CSH theoretical prediction models which are compared with the candidate biological GSs in an iteratively optimal way [(Equation 1 described below after the Gold Standard) that balances the recall (sensitivity) and precision and the CSH theoretical prediction model with the largest F-score is selected. Here we present, as an example, our procedure for identifying mouse probes that are most likely to hybridize with human RNAs, the converse would follow similar steps.To predict human-mouse cross-hybridizations and further redesign the probes, we optimize both the statistical models for biological measurement , are determined using the ideal F-Score while systematically varying each parameter independently .• dization   and F-scores (F) were calculated for each theoretical model of cross-hybridization against each biological GS at different thresholds of biologically cross-hybridizing probes (Equation 1).• Identification of cross-hybridizing probes (Table ): Once the optimal set of BLAST prediction parameters and biological GSs were determined, all probes from the GS that were considered as CSH were tagged and new SSPs were designed - regardless of whether they were predicted as cross-hybridizing by BLAST. Additionally, probes were tagged for future design when they did not have any expression on the array and were predicted as cross-hybridising using BLASTn program (Table • es Table : Once thI. Gene targets associated with cross species hybridizing probes were identified for redesign. Transcripts (RefSeq IDs) associated with each probe were identified from the Agilent microarray annotation file . For those probes with a retired Refseq or Unigene ID, the batch BLASTn program [ program  was usedII. New probes for the identified gene targets were designed using the "GE Probe Design" program in the Agilent eArray  software. The eArray parameters, that we used, include probe length (60nt), probes per target (1), probe orientation (sense) and design options ("best probe methodology" and "design with 3' bias"). These probes were further verified computationally for cross-hybridization.III. Newly designed probes were evaluated by the eArray "Probe Check" program using the built-in transcriptome. The new candidate probes were retained if they did not hybridize within their species with other targets than the intended one nor across species with any other transcripts. Otherwise, these candidate probes were tagged as inappropriate and new probes were designed for the intended target iteratively until they met these criteria.IV. All probes of the commercial arrays were retained, while new probes were also added to the H&M array. Annotation tables of cross species hybridization were used as filters to identify the most SSP for each gene, while the legacy probes were intended to allow for comparison of results with studies using the commercial platforms.Dataset S1). This extensive annotation of CSH was conducted by systematically extending the threshold of the biological GS in 10% increments from 0% to 100% for all probes.To validate the newly designed probes and extensively annotate the H&M array version 2, a second biological experiment was conducted by hybridizing all the probes on the H&M array version 2 to three human melanoma cell lines (MDA-MB-435) and three universal mouse mRNAs. Six Samples (three with human RNA and three with mouse RNA) were hybridized to the H&M array version 2.0 Table . The subHousekeeping genes are typically constitutively expressed genes required for the maintenance of basal cellular functions, and are thus assumed to be stably expressed under the same experimental conditions as well as between human tissues and mouse tissues ,23. To eFor simplicity, a set of probes classified as "species-specific" in the 2nd criteria that passed the threshold x are hereafter named "xSSP ". Different thresholds were tested, and the optimal threshold was selected according to the lowest variance of resulting RNA ratios.The SSPs satisfied the following four criteria: 1) they were positively expressed  across all samples when exposed to the intended RNA, 2) among all probes for one species, they expressed below a certain percentage of all expression values when exposed to the RNA of the other species, 3) they were not identified as CSH probes, and 4) were not theoretically predicted as CSH probes by the BLASTn algorithm. in vitro biological experiments data for which the proportion of mouse to human RNA is designed and known (GSE23377) were used to validate the mouse to human RNA ratio metric. This metric then serves as a proxy for stroma to cancer cell RNA ratio in xenograft models consisting of human cancer cell tumors harvested from mice hosts.Evaluation of the stromal cell to cancer cell mRNA ratio, hereafter named as S/C ratio for simplicity, was independently carried in two steps. The samples of the GOStats package[hexbin with default parameters.After masking the top 5% of the most likely probes to exhibit CSH, KEGG/GO enrichment analysis was performed among the genes targeted by probes on the custom array using the conditional hypergeometric test in the Bioconductor s package. For coms package and paths package. HexagonOur previous study reported interspecies genetic differences and elucidated stromal microenvironment signals from probes on human arrays unintentionally cross-hybridizing with mouse homologous genes in xenograft tumor models . By ideMethods, 5% threshold CSH), we identified 4,259 commercial probes as cross-hybridizing  corresponding to 3,550 distinct genes. Therefore, we designed a custom 2x105k array  combining all probes from both the human and mouse Agilent 44k commercial whole genome arrays . In addition, the H&M array contains new species-specific probes we designed: 5.5k human and 5.2k mouse , covering 30k distinct human targets and 32k mouse targets (Refseq IDs), respectively.Using our computational cross-species hybridization algorithms and the initial biologic experiment (- Supp. Table S2). Annotation for version 1 of the array was conducted by comparing the results from the initial in vitro biological experiment with the CSH theoretical predictions of probes on the H&M array. The optimal biological GS of CSH as observed in the in vitro experiments was defined by comparing the optimal CSH theoretical prediction model with the most ideal parameters using a threshold of 18% for mouse probes [A total of 87k probes were included on the H&M platform version 1, containing all commercial human and mouse probes, and 3,608 newly custom designed probes , where - Supp. Table S2, H&M array version 2 included 7.1k new custom probes, containing 5k probes designed from experimentally annotated CSH probes in version 1 of the platform, in addition to 2.1k probes that the Agilent protocol suggested as acceptable but whose theoretical prediction resulted in CSH. Together with the 3.6k custom probes designed in version1 array, the total 10.7k custom probes covered 16% (5.3k) of human genes and 15% (5.1k) of mouse genes . The set of BLAST prediction parameters derived from the initial in vitro biological experiment  performed better in predicting CSH probes than a similar set of parameters with shorter mismatch lengths . Two GSs for CSH (Table in vitro experiment [1- Supp. Table S2). This threshold of 5% was derived from H&M array version 1 probes, as version 2 included all version 1 probes. The estimated recall for mouse CSH probes was 25.6%  and precision was 13% , while the annotated recall and precision was 27% and 11%, respectively, for human CSH probes. Additionally, our extensive annotation stratified all probes on H&M array version 2 into ten levels according to their probability of CSH, thus providing array users with more flexibility to disregard CSH probes in their analyses.We further sought to incorporate theoretically predicted and experimentally verified CSH probes to the array. As illustrated in Figure 1periment , making panels C-D). Additionally, version 1 annotated CSH probes were significantly over-represented among the version 2 annotated CSH probes . Here, the overlap between the annotated CSH probes demonstrates the robustness of our design to identify the CSH probes in a xenograft model.The reduction in CSH was further shown by an analyses illustrated in Figure in vitro validation experiment for stromal to cancer cell ratio estimation, H&M arrays were separately exposed to human and mouse RNAs , and as shown in Panel B, the expression of mouse probes exposed to only mouse RNA was larger than that when exposed to only human RNA (median ratio between mouse and human probes is 9.7). Changing the thresholds to define an individual probe as species-specific  to include from 10 to 50 pairs of human and mouse SSPs provided a similar result, however the expression ratio between human and mouse probes tended to be unchanged when all non-SSP were included (data not shown). These results suggested that the proportion of each species RNA can be estimated by the species-specific human and mouse probes of the pair-wise homologous housekeeping genes.As shown in Figure in vitro biological experiment, where the stromal cell proportions were controlled using known quantity of RNAs as shown in Table in vitro CSH experimental data, where two arrays containing both human and mouse probes were exposed to equal quantities of human and mouse RNAs (GSE23377), the ratios of 13 pairs of homologous housekeeping gene expression were larger than 10-fold when exposed to the intended RNA of one species only , while was between 0 and 10 when exposed to an equal mix of human and mouse RNAs simultaneously  significantly (FDR <5%) over-represented among the 44,144 human SSPs . We found no bias in GO or KEGG for the mouse probes of the custom H&M array. Notably, gene ontology and pathway biases exist among the CSH probes on commercial single-species arrays, and should be accounted for on-going and future xenograft tumor studies. Here we are the first to report comprehensive analysis of CSH probes between human cancer cells avoiding these biases. We identified eight significantly enriched (FDR<5%) KEGG pathways , eight GO molecular functions , and conclusively, stromal functions detected when using a dual species array for xenograft tissues due to gene expression in mouse stromal tissues, rather than in human cancer cells (data not shown). Further, CSH probes confound the results of microarray analyses of the metabolism pathway of xenobiotics by cytochrome P450 when xenograft tumor models were used due to the highly conserved nature of many cytochrome P450 genes (data not shown). Using Agilent 44k human arrays, Sugawara et al. reported 28 genes demonstrating higher induction in xenograft tumors compared to the cells in culture medium[-5) in the CSH probes found in the list of 28 genes (data not shown), as would be expected for our xenograft model. Other significant pathways and functions listed that suggest relevant stromal functions include inflammatory signalling through the complement system, focal adhesion and tight junctions involving transmembrane proteins mediating intra-membrane and paracellular diffusion, as well as motor activity and actin binding, and thus possibly mediating invasion and metastasis processes. These results indicated that the design of dual-species arrays and the identification of CSH probes are critically important for studies involving highly conserved genes and pathways in xenograft models.To control for CSH of probes in GO and KEGG pathway enrichment analyses, we masked the 2,324 CSH human probes on the custom H&M array, and observed no KEGG pathways and only 1 GO molecular function . By design, we select a framework of well-established computational and biological methods to identify cross-hybridizing probes, construct a cross-hybridizing map, and identify new SSPs. Here, we provide a combined description of the interplay between the computational predictions of CSH of probes using BLASTn and observation of biological CSH that were utilized to develop an approach stratifying probes in terms of their propensities for cross-hybridization. We also implemented a rigorous rationale for the selection of a threshold needed to redesign the cross-hybridizing probes and offer the foundational datasets used for conducting analyses over a dual-species array: a comprehensive map of cross-hybridizations annotated with both sequence alignment and biological data intended to increase the accuracy and efficiency of measurements in tumor xenograft models. Additionally, we developed a novel method to estimate the mRNA ratio between mouse and human mRNA as a cellularity ratio, thus providing an alternative to conventional histological counts of stroma vs. cancer cells in xenograft models. Of note, the H&M array could also be utilized in single species experiments, as long as the probes of that species are selected. This information is clearly available in the "species" column of Dataset S1. Further, for comparison of H&M array results against previously published dataset using single species commercial Agilent arrays, the H&M probes corresponding to the commercial platforms are annotated in the column "designer" of Dataset S1.We established that overlooking CSH probes in xenograft models using commercial human arrays confound incorrect attribution of deregulated stromal genes to cancer cells. To increase the capability of analysing molecular changes in the stromal compartment as well as the interactions between cancer cells and reactive tumor stroma in xenograft modelling, we designed the first custom genome-wide human-mouse microarray, and biologically validated it by observing an overall reduction of the CSH  and two supplement figures . • Table S1 - Data resources used in this study. • Table S2 - Summary of probes on H&M array version 2 • Figure S1 - Optimization of Models identifying cross-species hybridizing probes. • Figure S2 - BLAST parameter selection based on higher F-scores.Click here for file


To reduce sampling error associated with cancer detection in prostate needle biopsies, we explored the possibility of using fluorescence in situ hybridisation (FISH) to detect chromosomal abnormalities in the histologically benign prostate tissue from patients with adenocarcinoma of prostate.MYC (8q24), LPL (8p21.22), and PTEN (10q23), as well as with centromere enumerator probes CEP8, CEP10, and CEP7. A distribution of FISH signals in the tumour and histologically benign adjacent tissue was compared to that in BPH specimens using receiver operating characteristic curve analysis.Tumour specimens from 33 radical prostatectomy (RP) cases, histologically benign tissue from 17 of the 33 RP cases, and 26 benign prostatic hyperplasia (BPH) control cases were evaluated with Locus Specific Identifier (LSI) probes MYC gain, CEP8 Abnormal, PTEN loss or chromosome 7 aneusomy was positive in the tumour area of all of the 33 specimens from patients with adenocarcinomas, and in 88% of adjacent histologically benign regions (15 out of 17) but in only 15% (4 out of 26) of the benign prostatic hyperplasia control specimens.The combination of A panel of FISH markers may allow detection of genomic abnormalities that associate with adenocarcinoma in the field adjacent to and surrounding the tumour, and thus could potentially indicate the presence of cancer in the specimen even if the cancer focus itself was missed by biopsy and histology review. Prostate carcinoma is the most common type of cancer in men in the United States, with an estimated 241,740 new cases in 2012 and 28,170 deaths ,4. ThereThe concept of “field cancerisation” or “field effect” was first proposed by Slaughter et al. in 1953 when observing histological features of oral cancers  to descrProstate cancer is multifocal disease, and field effect may play a fundamental role in the development of multifocal lesions. A recent review by Trujillo KA et.al  summarizMYC) locus, and loss of 8p21-22 (LPL) [PTEN), among others [LPL, MYC, PTEN, CEP7, CEP 8, and CEP 10. In this study, using FISH technique, we assessed whether these biomarkers could detect chromosomal abnormalities that are present in the histologically benign region adjacent to frank carcinoma.Although Florescence In Situ Hybridization technique (FISH) represents a molecular technique that allows the detection of numerical and structural genomic abnormalities in interphase cell nuclei in tissue sections or cytological specimens such as deletion, amplification, and translocation of various genomic regions in many types of cancer ,17, it hPTEN (SpectrumOrange™), MYC 8q24 (SpectrumGreen), and LPL 8p21-22 (SpectrumOrange). All probes were obtained from Abbott Molecular, Inc. .A total of 6 probes including 3 centromeric probes (CEP®) and 3 locus-specific identifiers (LSI®) were used. CEP probes included CEP7 (SpectrumAqua™), CEP8 (SpectrumAqua), and CEP10 (SpectrumGreen™). LSI probes were Thirty-three archived RP cases from patients with prostate adenocarcinoma and 26 control Benign Prostatic Hyperplasia (BPH) cases were provided by Rush University Medical Center (Approved IRB L06052503 waived the requirement for informed consent). Multiple tissue blocks were prepared from each of the RP cases. For each specimen, 5 μm tissue sections were cut and placed on positively charged microscope slides. The blocks were characterised by staining one out of 10 serial sections through the block with haematoxylin and eosin (H&E) followed by examination by an expert pathologist. For all 33 cases, at least one block containing adenocarcinoma was identified for this study and designated as “tumour”. Region(s) with histopathological features of adenocarcinoma were marked by the pathologist on the H&E slides of the tumour specimens. Twenty-five of the 33 adenocarcinoma cases were determined to have a Gleason score of 5–7, 6 cases had a Gleason score of >7, and 2 had a Gleason score of 2–4. For 17 out of 33 adenocarcinoma cases used in this study, a second block was identified that contained no recognisable histological features of adenocarcinoma or prostatic intraepithelial neoplasia (PIN) and designated as “histologically benign.” It was estimated that histologically benign tissue was spatially separated from the tumour margin on average by approximately 1 cm. The H&E images of the 17 histologically benign slides used in the study are provided in the Additional file The specimen slides used for the FISH assay procedure were within 10 serial sections of the respective H&E-stained slide to assure minimal separation of the areas examined by FISH from the areas evaluated by histopathology.MYC 8q24 (SpectrumGreen), and LPL 8p21-22 (SpectrumOrange); Probe mix 2 consisted of CEP7 (SpectrumAqua), CEP10 (SpectrumGreen), and PTEN (SpectrumOrange). Ten microliters of either probe hybridisation mix containing blocking DNAs and LSI/WCP Hybridisation Buffer  were added to a specimen, and a coverslip was applied with rubber cement sealed around. Slides and probes were codenatured for 5 minutes at 73°C and hybridized for 16–24 hours at 37°C on a ThermoBrite® hybridisation platform . Following hybridisation, coverslips were removed by soaking the slides in 2× SSC/0.3% NP-40 for 2–5 minutes, and immediately slides were washed in 2× SSC/0.3% NP-40 at 73°C for 2 minutes and subsequently in 1× SSC solution, PH ~ 7.0 for 1 minute at room temperature. The slides were then allowed to dry in the dark. Ten microliters of 4',6-diamidino-2-phenylindole counterstain/antifade solution  was added to the specimen, and a coverslip was placed on the slide for microscopy.Formalin fixed paraffin embedded (FFPE) histological specimen slides were baked at 56°C for 2–24 hours, then were treated three times in Hemo-De (Scientific Safety Solvents) for 5 minutes each at room temperature followed by two 1-minute rinses in 100% ethanol at room temperature. Slides were incubated in pre-treatment solution  at 80°C for 35 minutes, rinsed for 3 minutes in deionized water, incubated 20–22 minutes in 0.15% pepsin in 0.1 N HCl solution at 37°C, and rinsed again for 3 minutes in deionized water. Slides then were dehydrated for 1 minute each in 70%, 85%, and 100% ethanol and air-dried. Two sets of probe hybridisation mix were made: Probe mix 1 included CEP8 (SpectrumAqua), The specimens were analysed under a fluorescence microscope using single bandpass filters  specific for DAPI, SpectrumOrange, SpectrumGreen, and SpectrumAqua. The number of FISH signals for each probe was recorded in a minimum of 50 consecutive non-overlapped, intact interphase nuclei in areas of interest, which were identified by DAPI staining of nuclei with reference to the corresponding H&E-stained tissue. Tumour areas  scribed by the pathologist were evaluated on the 33 RP specimen slides. For the 17 slides with histologically benign tissue adjacent to tumour, representative areas were evaluated . Similarly, representative areas were evaluated on BPH specimen slides.For each specimen, 50–100 cells were enumerated with respect to the number of fluorescent signals of each probe. The following FISH abnormality parameters were calculated for each probe:• %Gain, percent cells with > 2 signals• %Loss, percent cells with < 2 signals• %Abnormal, percent cells with > 2 or < 2 signals• For the two probe ratios (probe A/probe B), %Gain is the percentage of cells with A/B ratio >1, and %Loss is the percentage of cells with A/B ratio < 1.t-test. FISH parameters with significant p-values  from the t-test were selected for further examination.In order to screen for FISH probes potentially important for disease detection, the FISH parameters described above were compared between different specimen groups  using a two-sample After prioritizing potential FISH probes, the receiver operating characteristic (ROC) method  was usedDFI is defined as All analyses were performed using SAS version 9.2  on a UNIX operating system by Abbott Molecular Biostatistics and Data Management Group.MYC (8q24), LPL (8p21.22), PTEN (10q23), and p16 (9p21), centromere probes CEP8, CEP10, CEP7, CEP3, and CEP17, and TMPRSS2 break-apart. In the study, we observed that tumour ROIs in radical prostatectomy specimens bore chromosomal abnormalities, including MYC amplification/gain, LPL and PTEN loss, TMPRSS2 rearrangement, as well as general aneuploidy. Importantly, we also observed chromosomal abnormalities in some of the 9 histologically benign slides from tumour patients. The results demonstrated the feasibility of using a FISH assay to detect chromosome abnormalities that are specific to specimens from adenocarcinoma patients. Based on these initial results, we selected six probes that detected chromosomal copy number abnormalities in most RP specimens, both within tumour regions and extending beyond histologically evident tumour. The six probes included MYC, LPL, CEP8, PTEN, CEP10, and CEP7.In an initial feasibility study, we tested 16 RP specimens from patients with adenocarcinoma of the prostate. Nine out of the 16 specimens had a second section available with only histologically benign tissue. Slides from 11 BPH cases were used as controls. The probes included LSI We expanded the initial specimen set to the total of 33 RP specimens from patients with adenocarcinoma of prostate and 26 BPH control specimens for the interrogation with the six selected FISH probes. For 17 out of 33 adenocarcinoma cases, in addition to the tumour region, we also evaluated tumour-adjacent histologically benign tissue from the same specimen, using a separate FFPE block that contained no histological features of adenocarcinoma or prostatic intraepithelial neoplasia (PIN) upon histopathological examination.MYC, LPL and PTEN, and aneusomy (as measured by copy number changes of the chromosome-specific CEP probes) were observed in tumour ROIs of the radical prostatectomy specimens. Figure Chromosomal abnormalities of In this study, FISH analysis was performed on histological specimens. In contrast to cytology, FISH on FFPE tissue specimens presents artefacts related to the nuclear truncation. In our study, FISH analysis of individual signal counts showed a loss up to 10% of signals, with average counts of FISH probe signals per cell of 1.80-1.84 in both test (RP) and control (BPH) tissue specimens. Therefore, although truncation effects were evident, the level of truncation did not appear to differ between test and control cases. To further control for artefacts of nuclear truncation, the cut-offs for FISH positivity, especially for deletion probes, were chosen above the level of truncation, as presented in the section Data Analysis of Probe Performance below.t-test comparing tumour ROI and BPH. These parameters were CEP10%Abnormal, CEP10%Gain, CEP7%Abnormal, CEP7%Gain, CEP8%Abnormal, CEP8%Gain, CEP8%Loss, MYC%Gain, LPL%Abnormal, LPL%Loss, PTEN%Loss, PTEN/CEP10%Loss, CEP7/CEP10%Gain, LPL/CEP8%Loss, MYC/CEP8%Gain and MYC/LPL%Gain. Results from t-test analyses demonstrated that for all of the 16 FISH parameters, mean values were statistically different between tumour and BPH groups , a very close, detailed inspection by the independent pathologist revealed possible minute tumour foci, however, the pathologist was not able to conclusively classify the observed cells as tumour without a suggested confirmation by other methods (such as IHC), highlighting the challenges in histopathological assessment of prostate tissue specimens. Therefore, we confirmed that FISH detected cytogenetic abnormalities in the regions of prostate that would not have been identified as tumour on histopathology review.Results from t-tests comparing histologically benign ROI and BPH controls, showed that for 10 out of the 16 FISH parameters  analysis method was applied to the selected ten single FISH parameters. AUC values from the ROC analysis comparing histologically benign ROIs from adenocarcinoma RP specimens with BPH controls were used to assess the selected FISH parameters in respect to their ability to distinguished adenocarcinoma specimens from the BPH controls based on the presence of the genomic abnormalities extending beyond the tumour margin. Table The selected single FISH probe parameters listed in Table PTEN%loss > 33, CEP7%Abnormal > 28, MYC%gain > 35, and CEP8%Abnormal > 34. Using these cut-offs, probe combination 1 yielded a sensitivity of 88.2% and a specificity of 84.6% for histologically benign ROI vs. BPH (AUC = 0.917), while the sensitivity and specificity for tumour ROI vs. BPH were 100% and 84.6%, respectively, with the AUC = 0.960.As evident from Table PTEN%loss, CEP7%Abnormal, MYC%gain, CEP8%Abnormal, are plotted in Figure ROC curves for the selected 4- probe combination, and the corresponding 4 single FISH probe parameters, including Each year, millions of men are referred for prostate biopsies due to abnormal DRE or elevated serum PSA. Prostate biopsies are not only unpleasant, but also carry risks to the patient, and are expensive. Moreover, false-negative rates for initial prostate biopsies (particularly for sextant biopsies) are routinely reported to be between 10–25%, and repeat biopsies are essential components of prostate cancer detection . TherefoLPL, MYC, PTEN, CEP 7, CEP 8, and CEP 10 FISH probes, based on the published roles of these genes, and on our initial FISH study on FFPE prostate cancer and BPH specimens. Sixteen parameters were derived from the number of FISH signals at the above loci, and compared between prostate adenocarcinoma tumour tissue (tumour ROI) and BPH samples by the t-test. In this analysis, all 16 FISH parameters demonstrated a significant difference between the comparison groups, supporting the role of these genes in prostate cancer. In addition to the tumour ROIs, we found FISH abnormalities in histologically benign tissue separated from the tumour margin on average by approximately 1 cm .In this study, we tested The pattern of FISH abnormalities in the histologically benign ROIs was similar to that found in the corresponding tumour  that was superior in performance to that of individual probes (Table By combining the individual FISH parameters, we identified a probe combination (es Table . In the The results of our feasibility study of radical prostatectomy suggest that multi-colour FISH may find utility in detecting cytogenetic abnormalities associated with adenocarcinoma of prostate in the field around the tumour, and therefore could potentially aid in assessing negative biopsies of patients with suspected cancer. The hypothesis that FISH could be used to aid the detection of adenocarcinoma by assessing the tissue surrounding the tumour will be validated in the next phase of the investigation using prostate needle biopsy specimens.In this study of radical prostatectomy specimens, cytogenetic abnormalities were observed by FISH within regions of prostate adenocarcinoma, as well as within regions of benign histology extending beyond histologically evident tumour margin, indicating a field cancerisation effect in prostate cancer. Detection of field cancerisation by FISH may prove to have a utility in the evaluation of histology-negative biopsies from patients suspected of having prostate cancer, and therefore could aid histopathological evaluation by providing an indication of possible presence of malignancy. Although preliminary, the findings of a FISH panel with sensitivity >85% in histologically benign regions away from tumour and specificity ~85% in BPH, provide encouragement to pursue the utility of these cytogenetic markers further. Validation in a larger cohort of patients with both positive and negative biopsies is envisioned to confirm our findings.AUC: Area under the curve; BPH: Benign prostatic hyperplasia; CaP: Prostate cancer; CEP: Centromeric probes; DRE: Digital rectal examination; FFPE: Formalin fixed paraffin embedded; FISH: Fluorescence in situ hybridisation; LSI: Locus specific identifier; PSA: Prostate-specific antigen; ROC: Receiving operating characteristic; RP: Radical prostatectomy; ROI: Region of interest.Authors YZ, KP, LM have filed a pending patent application relating to the subject matter of this article, which patent application has been assigned to Abbott Molecular Inc.YZ analysed the histological samples, performed the statistical analysis, and drafted the manuscript. TP analysed the histological samples. BB analysed the histological samples. JD performed the statistical analysis and helped to draft the statistical section. PL performed the statistical analysis and helped to draft the statistical section. DE assisted in the appropriate specimen selection and sectioned the slides. JC provided clinical opinion, selected and provided appropriate specimens and helped conceive the study, and scribed the tumour region on the H&E slides. LM conceived the study and the study design, provided guidance on data analysis, KP coordinated the study and its design, analysed the histological samples, assisted in selection of probes, performed the statistical analysis, and helped draft the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/14/129/prepubH&E Images of the 5-μm sections of the 17 histologically benign specimens from the corresponding radical prostatectomy adenocarcinoma cases. For each case, the specimen slide used for the FISH analysis was within 10 serial sections of the H&E stained slide.Click here for filet-test analyses. Table S2a.t-test of tumour ROI vs BPH. Table S2b.t-test of histologically benign ROI vs BPH.Click here for fileThe pattern of FISH abnormalities presents in the 33 tumour ROIs and the 17 available corresponding histologically benign ROIs.Click here for file


T) on PPV and SVV, and prediction of fluid responsiveness in a model of intra-abdominal hypertension (IAH).Pulse pressure variation (PPV) and stroke volume variation (SVV) are frequently used to assess fluid responsiveness in critically ill patients on mechanical ventilation (MV). There are many factors, in addition to preload that influence the magnitude of these cyclic variations. We sought to investigate the effect of tidal volume (VT) of 6, 12 and 18 ml/kg before IAH after IAH induction and after a fluid challenge during IAH.Twelve anesthetized and mechanically ventilated piglets on continuous pulse contour cardiac output monitoring. Hypovolemia was ruled out with 2 consecutive fluid boluses after instrumentation. IAH was induced by intraperitoneal instillation of colloid solution with a goal of reducing respiratory system compliance by 50 %. Subjects were classified as fluid responders if stroke volume increased >15 % after each fluid challenge. SVV and PPV were recorded with tidal volumes  to 5 %  (p = 0.05), with 12 ml/kg from 5 %  to 11 %  (p = 0.02) and 18 ml/kg from 5 %  to 15 %  (p = 0.02). Similarly, PPV increased with all the tested VT after IAH induction, being this increase larger with high VT: with 6 ml/kg from 3 %  to 6 %  (p = 0.05), with 12 ml/kg from 5 %  to 13.5 %  (p = 0.02) and 18 ml/kg from 7 %  to 24 %  (p = 0.02). One third of subjects responded to fluid administration after IAH, but neither SVV nor PPV were able to identify the fluid responders with the tested VT.VT, but the magnitude of that change was higher with larger VT. This observation reveals the dependence of functional hemodynamic markers on intrathoracic as well intra-abdominal pressures, in addition to volemic status. Also, PPV and SVV were unable to predict fluid responsiveness after IAH induction. Future studies should take into consideration these findings when exploring relationships between dynamic preload indicators and fluid responsiveness during IAH.IAH induction in non-hypovolemic subjects significantly increased SVV and PPV with the three tested VThe online version of this article (doi:10.1186/s12871-015-0105-x) contains supplementary material, which is available to authorized users. Functional hemodynamic monitoring has been demonstrated to be a powerful tool in critically ill patients. Careful selection of patients that will respond to fluid administration may help avoid fluid overload. Functional hemodynamic monitoring including stroke volume variation (SVV) and pulse pressure variation (PPV), have been shown to be more accurate in predicting fluid responsiveness than classically used static parameters  and pulmonary artery occlusion pressure) in mechanically ventilated patients without spontaneous breathing –3.T), PEEP, driving pressure and respiratory rate (RR) as well as lung compliance are potential factors that may decrease the ability of functional hemodynamic monitoring parameters to predict fluid responsiveness [These dynamic indexes are based on cyclic transmission of airway pressure to the pleural and pericardial spaces, which induces changes in venous return and preload. Due to the complex relationship between intrathoracic structures (heart-lung interactions) numerous studies have demonstrated that functional hemodynamic monitoring parameters do not only depend on preload and ventricular interdependence. Mechanical ventilation (MV) settings like tidal volume  represents the final stage of a pathologic process caused by an increase in intra-abdominal pressure (IAP) to a degree that can compromise regional blood flow. It has significant morbidity, related to the ongoing inflammation due to ischemia and organ dysfunction, ultimately causing death , 8. JudiT, have been shown to modify functional hemodynamic monitoring parameters when lung compliance is reduced, like ARDS, due to low transmission of pressure from airway to pleura [T on functional hemodynamic monitoring in conditions of reduced respiratory system compliance due to extrapulmonary causes has not been studied. We sought to investigate the effect of different VT on PPV and SVV in a model of intra-abdominal hypertension. We hypothesize that acute increase in IAP increases SVV and PPV readings in euvolemic animals, and this increase is proportional to the size of VT.Cyclic changes in SV and PP induced by positive pressure ventilation can be affected by extra thoracic modifications in compliance, such as ACS or contained laparotomy , 16. Curo pleura , 27. HowThe experimental protocol was approved by Facultad de Medicina Clínica Alemana-Universidad del Desarrollo Ethics Committee and the CONICYT  Bioethics Advisory Committee. All of the experimental procedures were consistent with the Guiding Principles in the Care and Use of Laboratory Animals adopted by the American Physiological Society.T 10 ml/kg, PEEP 5 cmH2O, respiratory rate (RR) 20 breaths per minute (bpm), inspiratory time 0.75 s, and FiO2 0.5. Anesthesia and neuromuscular blockade were maintained by continuous infusion of propofol (10 mg/kg/h), fentanyl (4 μg/kg/h), and pancuronium (0.2 mg/kg/h) throughout the experiment. Hydration was maintained with a continuous infusion of normal saline at 5 ml/kg/h. The temperature was maintained at 37.2 ± 0.4 °C by conventional convective methods.12 anesthetized Large-White piglets (4.9 ± 0.05 kg) were used in this study. Animals were premedicated with intramuscular acepromazine (1.1 mg/kg) and ketamine (20 mg/kg). The trachea was cannulated with a 3.5-mm  cuffed tracheostomy tube , the left jugular vein with a 4 F double lumen catheter  and the right femoral artery with a 4 F thermistor-tipped catheter , all via cut down. A peritoneal dialysis catheter was inserted with Seldinger Technique under aseptic conditions. Animals were ventilated in a volume control mode . Initial settings were VT 6, 12 and 18 ml/kg in random order during the following experimental steps: baseline, after a 20 ml/kg normal saline (NS) fluid bolus (Fluid Bolus 1), after a second 20 ml/kg NS fluid bolus (Fluid Bolus 2), after intra-abdominal hypertension (IAH) induction and after a fluid bolus of 20 ml/kg during IAH (Fluid Bolus 3) . After testing the 3 different VT, subjects were placed on VT 10 ml/kg until next randomization. After each fluid challenge PiCCO® plus device was re-calibrated and all measurements were obtained after 15 min of stable hemodynamic conditions.Subjects were exposed to three different VRS) by 50 %. We chose this target to ensure that the experimentally induced IAP was high enough to induce significant extra-abdominal organ dysfunction. At the end of the experiment the animals were euthanized.IAH was induced by intraperitoneal instillation of Voluven® 6 %  in successive aliquots of 100 ml through the peritoneal catheter. Target IAP was defined as the IAP necessary to decrease the static respiratory system compliance  from blood samples drawn from the arterial catheter. Oxygenation was assessed with the PaO2/FiO2 ratio. CRS was calculated as VT/(Ppl - PEEPTOT), where Ppl is the plateau pressure measured after an inspiratory hold of 4 s, and PEEPTOT is the total end-expiratory airway pressure measured after an expiratory hold of 4 s. These variables were recorded from the ventilator display.Hemodynamic measurements: Heart rate (HR), mean arterial pressure (MAP), and central venous pressure were monitored with an Infinity® Delta XL monitor . Zero pressure was set at the midaxillary line. Cardiac index (CI) was measured with a commercially available device PiCCO® plus. According to manufacturer´s instructions, single thermal indicator transpulmonary thermodilution was performed in triplicate by injection of a 5-ml bolus of iced normal saline solution into the superior cava vein through the jugular catheter. The CI was internally computed from an analysis of the thermodilution curve with a modified Stewart-Hamilton algorithm [2/3, where K = 0.112 for pigs.lgorithm . The bodFunctional Hemodynamic measurements: In the PiCCO® plus device, SVV was calculated from the mean values of four minimum and maximum stroke volumes averaged during the last 30 s (SVmean):PPV was calculated during the same time interval:Fluid responsiveness: At the normal intra-abdominal stage, and after IAH induction, subjects were classified as fluid responders if SVI increased greater than 15 % after the fluid bolus [id bolus .Hemodynamic and respiratory measurements were performed at baseline and at the end of the experiment.U test was used to compare data between groups. Spearman rank correlations were performed between functional hemodynamic markers and changes in SVI. Significant outliers were defined as any value more than 1.5 × interquartile range and they were excluded of the analysis.Data are presented as median and 25th and 75th percentile range (P25–75), unless stated. Related-Samples Wilcoxon Signed Rank test was used to assess differences between baseline data and measurements at the end of the study and Independent-Samples Mann–Whitney T [Receiver-operating characteristic (ROC) curves were constructed to evaluate the capacity of PPV and SVV to predict fluid responsiveness under the three tested VT .P <0.05 were considered statistically significant. All statistical analyses were performed with the SPSS 20.0 software program .Differences with All the animals completed the experimental protocol. The hemodynamic data during the experiment are presented in Table T 12 and 18 ml/kg versus 6 ml/kg. These differences were attenuated after fluid loading , VT 12 ml/kg  and 18 ml/kg . Also a positive correlation was found between ΔSVI and PPV with VT 6 ml/kg , VT 12 ml/kg  and 18 ml/kg . Scatter plot of these correlations are provided as supplemental material  was achieved after intraperitoneal infusion of 2.5  l of colloid solution over 35  min. As planned in the experimental design, the rise of IAP caused a decrease in CRS from 1.27  to 0.55  ml/cmH2O/kg (p <0.01) and PaO2/FiO2 ratio from 412  to 330  (p <0.01). After IAH induction there was a significant decrease in CI with no changes in CVP and HR  to 5 %  (p = 0.049), with 12 ml/kg from 5 %  to 11 %  (p = 0.02) and 18 ml/kg from 5 %  to 15 %  (p = 0.02)  to 6 %  (p = 0.047), with 12 ml/kg from 5 %  to 13.5 %  (p = 0.02) and 18 ml/kg from 7 %  to 24 %  (p = 0.02)  mmHg at baseline. The IAP of 22 19, 23) mmHg  , VT 12 ml/kg  and 18 ml/kg   v/s 3  and PPV 5  v/s 3  for responders and non-responders respectively. In accordance to previous observations, airway pressurization was not enough to demonstrate preload dependency with low VT in mechanically ventilated healthy piglets [T 12 and 18 ml/kg. PPV had better performance predicting fluid responsiveness with all the tested VT, being better with 12 ml/kg with a cut off of 9.5, strikingly similar to previous studies [Forty two percent of subjects were fluid responders before IAH. Both PPV and SVV were higher in fluid responders compared to non-responders, but these differences were smaller with lower V piglets , 33. On  studies . Also IAPPV 5 3,  v/s 3  changes in SVV and PPV after IAH induction were very small and most animals were below the usual threshold described to predict fluid responsiveness. On the other hand, high VT produced a large increase in SVV and PPV, showing that during IAH in addition to the pressure of the abdominal compartment, PP and SV are dependent on the airway pressure swings, and not the applied VT. This supports the clinical observation of Muller et al. who found that lack of significant variation of airway pressure, expressed as PIP-Ppl, in patient on mechanical ventilation with low VT might limit the use of PPV to predict fluid responsiveness [We found that after IAH induction, PPV and SVV increased with the 3 tested Vpartment , 21, 28.siveness .T. It is important to note that AUC might look similar between pre-IAH and after IAH induction, but the wide confidence interval (crossing 50 %) as well as the large p-value observed, reflect that SVV and PPV did not discriminate between animals that increased their SVI greater than 15 % after the third fluid challenge. Other studies have found that SVV and PPV were useful to predict fluid responsiveness, but with different thresholds [T 12 and 18 ml/kg even in the absence of changes in SVI during fluid challenge can be explained by the large variation in intrathoracic pressures due to decreased CRS in accordance with our definition of IAH.Fluid administration during IAH increased SVI greater than 15 % in 1/3 of the subjects. There was a non-significant trend towards higher PPV and SVV in these fluid responsive patients. ROC analysis showed that PPV and SVV were not able to discriminate between fluid responders and non-responders during IAH with the tested Vresholds , 17, 19.T, airway pressurization and IAP when interpreting functional hemodynamic monitoring parameters to decide fluid administration.The present study should not be understood as challenging the ability of PPV or SVV to predict fluid responsiveness. With our initial hypothesis and the obtained results we intend to point out the relationship between abdominal and thoracic compartment, considering the VOur study has some limitations. First, in addition to the interspecies physiologic differences , we stuT at baseline and after IAH induction. These differences were attenuated after fluid administration. IAH induction in non-hypovolemic subjects significantly increased SVV and PPV with the three tested VT, but the magnitude of that change was higher with larger VT. This observation reveals the dependence of functional hemodynamic markers on intrathoracic as well intra-abdominal pressures, in addition to volemic status. In agreement with this physiological description, we found that PPV and SVV were good predictors of fluid responsiveness at baseline, but not after IAH induction, showing the complex relationship of the thoracic and abdominal compartments. Future studies should take into consideration these findings when exploring relationships between functional hemodynamic monitoring and fluid responsiveness in IAH.We found significant differences in PPV and SVV with different VAlso our observations need to be studied in other hemodynamic conditions, like hypovolemia, low cardiac output and sepsis, and should include measurements of pleural pressure, chest and abdominal wall compliance, for better understanding of determinants of the dynamic markers of fluid responsiveness.


Delayed bathing and early initiation of breastfeeding are among the essential interventions recommended to save newborn lives. Although survey coverage reports are key to monitoring these interventions, few studies investigated whether such reports accurately reflect the proportion of mothers and children who received these interventions. In order to gather accurate data, guidance on how to interview and probe mothers is provided. In this study, we investigated experiences of data collectors when asking mothers survey questions that assessed delayed bathing and early initiation of breastfeeding.In November 2013, using a self-administered semi-structured questionnaire, we interviewed data collectors who had taken part in a population-based newborn health household survey in Ethiopia during October-November 2013. A total of 130 out of 160 invited data collectors completed and returned the self-administered questionnaire. Descriptive statistics were used to analyse quantitative data using SPSS software version 19. Qualitative data showing the variety of probes used by data collectors was analysed by listing, screening to identify common themes, and grouping by category.Most data collectors reported that, in their opinion, mothers were able to understand the meaning of the question about newborn bathing  and breastfeeding initiation  without the need for probes. However, fewer mothers were able to recall the event for either newborn behaviours and describe it in minutes, hours or days without the need for probes. Overall, only 26% (n = 34) and 34 % (n = 44) of all data collectors reported that they did not need any probing for the questions related to newborn bathing and breastfeeding initiation questions, respectively. We identified a variety of probes used by data collectors and present examples.Considerable probing was necessary to facilitate maternal recall of the events and approximate their responses of time regardless of mothers’ age, level of education and parity. This could potentially lead to inaccurate coverage reports due to subjective and inconsistent interpretation of the indicators. Therefore, we recommend inclusion of standard probes or follow-on questions to the existing survey tools assessing the two indicators. Data collectors also require further guidance in using appropriate probes to gather accurate maternal responses.The online version of this article (doi:10.1186/s12887-015-0350-7) contains supplementary material, which is available to authorized users. Essential interventions immediately after birth such as thermal care (immediate drying and delayed bathing), early initiation of breastfeeding (within one hour) as well as hygienic cord and skin care are recommended to save newborn lives -3 and thAccurate measurement of essential interventions, including delayed bathing and early initiation, could inform policy makers in tracking progress made in saving newborn lives and permit comparison of coverage figures within and between countries. In many high mortality settings, tracking the extent to which every newborn benefits from these care components is limited by the lack of well-established routine health information systems. Population-based household surveys such as the Demographic and Health Survey (DHS) and the Multiple Indicator Cluster Survey (MICS) remain the main source of coverage reports based on which national and regional newborn health policies and strategic decisions are made. However, few studies have evaluated validity and reliability of such household surveys and it is unclear whether survey reports accurately reflect the proportion of mothers and children who accessed and received life-saving interventions .A recent review  identifiWe propose another challenge related to measurement of immediate newborn care behaviours and practices, which is the experience of the data collectors themselves. In order to gather accurate data, guidance on how to interview and probe mothers is provided to enumerators prior to data collection ,10. In tData were collected by inviting a large team of household survey interviewers, who were still in the field, to complete a self-administered semi-structured questionnaire and provide answers based on the most recent interview they had conducted with a mother of a newborn child.A population-based newborn health survey was conducted between October-November 2013 in four major regions of Ethiopia: Amhara, Oromiya, Tigray and Southern Nations, Nationalities and Peoples’ region (SNNP). The household survey, implemented as part of a measurement, learning and evaluation grant to the IDEAS project , at London School of Hygiene and Tropical Medicine, was designed to measure population level coverage of newborn behaviours and practices, including immediate breastfeeding and delayed bathing. The survey tools were modular, consistent with the DHS structure, and included a module for women aged 13–49 years who had a recent live birth. DHS tools include a coded response ‘immediately’ but this response was not included in this survey and the wording of the question assessing early initiation, shown in Table In order to minimize language and cultural barriers, data collectors were recruited from the same region where the interviews took place and all of them had college-level or above education in health and social sciences. In addition to participating an intensive training on the data collection tool and field guide, data collectors also participated in one-day pilot testing of the tool before they started actual interviews. A total of 160 data collectors were employed for the survey and all were invited to participate in this study sharing their experiences of interviewing eligible mothers.We developed a self-administered, semi-structured, paper-based questionnaire. An additional pdf file shows this in more detail ?”Most data collectors n = 102, 79%) reported that, in their opinion, mothers were able to understand the meaning of the question upon hearing it for the first time without the need for probes. However with regards to responses about timing of when the newborn was bathed, only 39% (n = 51) and 46% (n = 60) of data collectors reported that mothers were able to recall the event and describe it in minutes, hours or days without probing, respectively. There was evidence that a higher percentage of mothers who gave birth at home were able to give responses without requiring any probing compared to mothers with a health facility delivery . Overall, just over a quarter of all data collectors 26% (n = 34) reported that they did not need any probing to help mothers understand the meaning of the question, recall the event of newborn bathing, or describe it in minutes, hours or days  reported that, in their opinion, mothers understood the question about breastfeeding initiation upon hearing it for the first time without the need for probes. However, only 48% (n = 62) reported that they didn’t need to probe mothers both to help them recall the event of breastfeeding initiation or describe the time in minutes, hours or days. Overall, only a third of all data collectors 34% (n = 44) reported that they did not need any probing to help mothers understand the meaning of the question, recall the event of breastfeeding initiation or describe it in minutes, hours or days  probes which simply re-phrased the wording of the question, 2) probes that used some reference points during birth such as delivery of placenta, and 3) probes that sequentially listed all events surrounding birth in order to facilitate maternal recall.“From the time you gave birth, when was the first time the baby come in contact with water?” “As per the advice from health extension workers, did you delay bathing your baby to prevent baby getting cold and by how long?” “How many minutes or hours after (name) was born did you get her to breastfeed?” “Did he breastfeed as soon as you gave birth? Or did the baby breastfeed on the same day of his birth, or the second day, or the following consecutive days?” Examples of probes which simply re-phrased the wording or text description of the questions include the following.“How long after the placenta was delivered did you bath your baby?” “When you bath your baby for the first time after birth, was it breakfast time, lunch, dinner or time when the cattle came back home?” “When did you start breastfeeding your baby? Was it before the placenta came out or after? If it is, how long after delivery of the placenta did you start breastfeeding?” “Did you start breastfeeding after the placenta came out and you were given kacha ? How long after birth was that?” Probes which used reference points of an event around birth in order to help mothers recall the event and describe the time include the following.“What happened after the baby was born and placenta delivered? Did they dry the baby and wrap him in a cloth? What time did they bath him?” “Who attended the birth? Did they bath your baby? Where was the baby kept after that and when did they give you your baby to start breastfeeding?” “Did they bath your baby at the health center or was it after you went back home? How long did you stay in the health center after giving birth? Did you start breastfeeding in the health center or once you got back home? How long after birth could this be?” Finally, probes which were used to ask mothers sequentially going through all events following birth in order to estimate approximate time for bathing the baby include the following.Although most data collectors reported that the majority of mothers they interviewed understood the meaning of the essential newborn care questions, considerable probing was still needed to facilitate recall and approximate responses of time regardless of maternal age, educational level, parity and place of delivery. We also identified variation in the types of probes that the data collectors used.A limitation of this study was whether data collectors accurately recalled and reported their experience of interviewing mothers using the self-administered questionnaires provided. Attempts were made to minimize recall bias, for example by asking them to base their answers on the most recent interviews, and by providing them information sheet and orientation in order to help them accurately complete the questionnaire. Nonetheless, data collectors might have chosen to share their experiences based on the “best or worst” interviewee they had and therefore, their perception of how mothers understood the questions could still be inaccurate.Despite this limitation, some consistent results emerged. According to data collector reports, although most mothers were clear about the wording or text descriptions of both questions, considerable probing was necessary to facilitate maternal recall of the event and approximate their responses of time as per the recommended coding format. Consistent with a study in South Africa , we founHowever, we did observe that mothers who gave birth at home could answer questions without probing more frequently than mothers who gave birth in a health facility. This could partly be explained by limited involvement of mothers with immediate newborn care practices due to hospital protocols even when they had non-emergency deliveries, and therefore mothers may need more prompting to remember the sequence of events. We also identified different examples of probes used as follow-on questions for mothers who needed clarification about the questions. Currently, there are no standard probes or follow-on questions recommended in household surveys such as DHS ,12 and MAccuracy in self-reports of mothers who participated in population-based household surveys have been assessed ,15 and rData collectors reported that considerable probing was necessary to facilitate maternal recall of the event and approximate their responses of time regardless of mothers’ age, level of education and parity. This could potentially lead to inaccurate coverage reports due to subjective and inconsistent interpretation of the indicators, which makes it difficult to track progress in the implementation of newborn life-saving interventions. Therefore, we recommend inclusion of standard probes or follow-on questions to the existing survey tools assessing the two newborn indicators. Data collectors also require further guidance about the purpose and interpretation of the indicators as well using appropriate probes to gather accurate maternal responses.The data set is published on LabArchives with DOI: 10.6070/H4SQ8XDD.