Puromycin is a tyrosyl-tRNA mimic that blocks translation by labeling and releasing elongating polypeptide chains from translating ribosomes. Puromycin has been used in molecular biology research for decades as a translation inhibitor. The development of puromycin antibodies and derivatized puromycin analogs has enabled the quantification of active translation in bulk and single-cell assays. More recently, in vivo puromycylation assays have become popular tools for localizing translating ribosomes in cells. These assays often use elongation inhibitors to purportedly inhibit the release of puromycin-labeled nascent peptides from ribosomes. Using in vitro and in vivo experiments in various eukaryotic systems, we demonstrate that, even in the presence of elongation inhibitors, puromycylated peptides are released and diffuse away from ribosomes. Puromycylation assays reveal subcellular sites, such as nuclei, where puromycylated peptides accumulate post-release and which do not necessarily coincide with sites of active translation. Our findings urge caution when interpreting puromycylation assays in vivo. Puromycin is a potent translational inhibitor that binds to ribosomes from all domains of life and has been used as a chemical probe and selectable marker for decades . PuromycThe development of anti-puromycin antibodies and of derivatized analogs of puromycin  has led In the present work, we establish that puromycin-based methods, as currently implemented, do not accurately localize translation at the subcellular level. We used a rabbit reticulocyte lysate system to show that puromycin nearly instantaneously releases nascent proteins from the ribosome, and that this release reaction is completely unaffected by emetine. To validate this finding in cells, we visualized sites of active translation using fixed cell single-molecule imaging with the SunTag reporter system. Brief treatment with puromycin nearly completely dissociated nascent peptides from their mRNAs, again, independent of the presence of emetine. Simple diffusion calculations predict that the released peptides could diffuse to nearly any point within even large mammalian cells within seconds or minutes. Thus, puromycylation methods described in the literature do not establish subcellular localization of translation.C. elegans gonads, we observed bright labeling of live germlines upon a 5 min incubation with OPP. The OPP signal was most intense in nuclei, specifically in the chromatin-free center where nucleoli reside. A lower signal was also observed in the cytoplasm, which contains the majority of  functional ribosomes (O-propargyl-puromycin (OPP) is a click-reactive cell permeable puromycin analog that is commonly used to localize sites of translation . When inibosomes . OPP labibosomes , therebyibosomes . Emetineibosomes  and may ibosomes . We concTo determine whether emetine prevents release of puromycylated nascent peptides in vitro, we made use of a previously established real-time translation monitoring assay in rabbit reticulocyte lysate (RRL). This method relies on the fact that luciferase rapidly folds into an enzymatically active conformation only after release from the ribosome . By progWe reasoned that if emetine prevents release of puromycylated nascent peptides, then emetine should block the increase in luminescence observed upon puromycin addition. Matching the conditions of We next considered the possibility that blocking peptide release with emetine requires pre-incubation. To test this, we pre-treated the lysate with emetine 5 min before addition of puromycin. Because pre-treatment would decrease the total translation time and overall luminescence of a sample, it was critical to equalize the total uninhibited reaction time of all samples. This was accomplished in two different ways. In a first experiment, we treated all samples with the nonhydrolyzable GTP analog 5'-guanylyl imidodiphosphate (GDPNP) to inhibit the translational GTPases and prevent ongoing translation while leaving the ribosome free to react with emetine and puromycin . Again, To directly test whether emetine blocks release of puromycylated nascent chains in vivo, we implemented the SunTag method for monitoring translation on single mRNAs . This teIn untreated cells, an average of 63% of single mRNAs (red foci) per cell co-localize with bright SunTag signal (green foci) . Upon trWhile initial reports argued that emetine was required to stabilize the interaction of puromycylated peptides with ribosomes, some recent studies of local protein synthesis via the puromycylation method relied on treatment with puromycin alone for ~5–10 min, with the implication that detected nascent proteins do not appreciably diffuse away from their site of synthesis (i.e. ribosome) within the treatment time . To deteIn this work, we have demonstrated that the puromycin method for visualizing localized translation does not faithfully detect nascent polypeptides at the site of their synthesis. Puromycylated polypeptides are released from the ribosome and can diffuse far away from the site of synthesis, even following short treatment times. Additionally, treatment with emetine does not prevent release of puromycylated peptides from the ribosome. This is in agreement with previous polysome gradient analysis  and realA Kozak consensus sequence (GCCACC) was inserted immediately upstream of the start codon of luciferase reporter plasmid pGEM-luc (GenBank X65316.2) to generate pSL312. This was used as the template for full-length firefly luciferase mRNA transcription and was linearized with a StuI restriction digest. For reasons unrelated to the current work, a disabled 2A peptide sequence was fused downstream of the luciferase sequence to generate P3.28_pGEM_ luc_2A_AGP|_kozak_RC, which was further modified by inserting an HpaI restriction site 2 nt 3′ of the final codon of luciferase  through site-directed mutagenesis to make P3.35_pGEM_luc_trunc_kozak_RC. This plasmid was linearized with an HpaI restriction digest such that transcription of this template would terminate at TTGtt and would exclude the 2A peptide sequence. For SunTag experiments, the plasmid pcDNA_CMV_ST was used to generate a stable cell line using the Flp-In method. pcDNA_CMV_ST contains an open reading frame coding for 23x SunTag repeats, Nano Luciferase, BFP and an auxin-inducible degron, expressed from a pcDNA5 vector.C. elegans was cultured according to standard methods at 20 °C. N2 adult germlines were dissected into egg buffer with 1 mM levamisole. Germlines were incubated with 45 µM emetine, or 37 µM anisomycin  and Slidebook v6.0 software (Intelligent Imaging Innovations). Germline images are 10 μm z-stacks starting at the bottom of the distal germline with 0.27 μm step size using a 63X objective. Average intensity projections were quantified in ImageJ. An ROI was drawn around the mitotic zone of each germline and fluorescence in the 488 nm channel was measured. Fluorescence intensity of each germline was normalized to the average intensity of the germlines treated with OPP alone.Luciferase plasmids were linearized with a blunt-end restriction enzyme just upstream (truncated) or downstream (full-length) of the stop codon, followed by transcription with the mMESSAGE mMACHINE SP6 transcription kit (Invitrogen AM1340). Synthesized mRNA was quantified using a Nanodrop 1000. Nuclease-treated rabbit reticulocyte lysate translation reactions (Promega L4960) were set up in a 384-well plate (Thermo Scientific 164610) on ice. Luciferin (PerkinElmer 122799) was added to each reaction well to a concentration of 0.5 mM followed by 12 units of Superase-In RNase Inhibitor (Invitrogen AM2696). SP6-transcribed truncated or full-length firefly luciferase mRNA was added to a concentration of 40 µg/mL using a multichannel pipette and the plate was immediately inserted into a luminometer microplate reader (Biotek Synergy H1MD) regulated at 30°C. Luminescence readings were taken every few seconds, depending on the number of reaction wells. 5'-guanylyl imidodiphosphate  was added to the wells 16 min after the start of the reaction for 5 min at a concentration of 100 µM followed by a 5-min pretreatment of either 208 µM emetine  or 9.4 µM anisomycin (Sigma A9789). Puromycin (Sigma Aldrich P7255) was added to wells at a concentration of 91 µM. In experiments where GDPNP was not used, the first translation inhibitors were added to the reaction wells at 21 min following the start of the reaction. Reagents were added to the wells by first ejecting the microplate from the luminometer and pipetting the reagents in using a multichannel pipette. The microplate was then promptly inserted again.U-2OS cells stably expressing the SunTag reporter were generated using the Flp-In system with the pcDNA_CMV_ST plasmid, as described in smFISH-IF smFISH-IF was performed similarly as described . smFISH-E. coli tRNA (Sigma-Aldrich 10109541001), 10% w/v dextran sulfate (Sigma-Aldrich D8906-100G), 2 mM ribonucleoside vanadyl complex (NEB S1402S), 100 units/mL SUPERase In (Thermo Fisher AM2694), 60 nM SunTag_v4-Cy3 smFISH probes, and 1:1000 chicken anti-GFP (Aves Labs GFP-1010). The coverslips were then washed 4x with 2xSSC (Corning 46–020 CM) + 10% formamide (Sigma-Aldrich F9037-100ML). The samples were then incubated with 2 × 20 min with a goat anti-chicken IgY secondary antibody labeled with Alexa Fluor 488 (Thermo Fisher A-11039). After 3x washes in 2xSCC, cells were mounted on pre-cleaned frosted glass cover slides (Fisher 12-552-3) with ProLong Diamond antifade reagent with DAPI (Invitrogen P36962). After curing for 24 hr, the samples were imaged on a custom Nikon Ti-2 wide-field microscope equipped with a 60 × 1.4 NA oil immersion objective lens (Nikon), a Spectra X LED light engine (Lumencor), and an Orca 4.0 v2 scMOS camera (Hamamatsu). The microscope was under automated control by Nikon Elements software. x-y pixel size: 107.5 nm. z-step: 300 nm.Approximately, 24 hr following induction, cells were treated with either 91 µM puromycin in the medium for 5 min, 208 µM emetine in the medium for 15 min followed by 91 µM puromycin in the medium for 5 min, or 37 µM anisomycin in the medium for 5 min followed by 91 µM puromycin in the medium for 5 min. Control cells were left untreated. Following treatment, samples were prepared for smFISH-IF. All solutions were prepared in nuclease free water . Cells were washed 3x with 1x PBS (Corning 46–013 CM) + 5 mM magnesium chloride (Sigma-Aldrich M2670-500G) (PBSM). Cell were then fixed for 10 min at room temperature in PBSM + 4% paraformaldehyde (Electron Microscopy Sciences 50-980-492). Following fixation, samples were washed for 3 × 5 min in PBSM and permeabilized for 10 min in PBSM + 5 mg/mL BSA (VWR VWRV0332-25G) + 0.1% Triton-X100 (Sigma-Aldrich T8787-100mL). After 3 × 5 min washes in PBSM, cells were incubated for 30 min at room temperature in 2xSSC (Corning 46–020 CM), 10% formamide (Sigma-Aldrich F9037-100ML), and 5 mg/mL BSA (VWR VWRV0332-25G). Following pre-hybridization incubation, samples were incubated for 3 hr at 37°C in 2xSSC (Corning 46–020 CM), 10% formamide (Sigma-Aldrich F9037-100ML), 1 mg/mL competitor Fixed cell image analysis was performed as described  with exi In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:The work convincingly shows that puromycin releases nascent polypeptides even in the presence of elongation inhibitors. This observation questions interpretations using detection of puromycin to denote sites of translation.Decision letter after peer review:eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by David Ron as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Marvin Tanenbaum (Reviewer #2); Maria Ugalde (Reviewer #3).Thank you for submitting your article "Puromycin reactivity does not accurately localize translation at the subcellular level" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.eLife papers without additional data, even if they feel that they would make the manuscript stronger. Thus the revisions requested below only address clarity and presentation.We would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). Specifically, we are asking editors to accept without delay manuscripts, like yours, that they judge can stand as Summary:The work presented here convincingly shows that the puromycylated signal comes from released, truncated polypeptides, and not the nascent chains associated with the ribosome. In a straightforward experiment, using the SunTag system, the puromycylated nascent chains were not present on ribosomes, but only after the peptides were released from the ribosome did the fluorescence of the tagged puromycylated peptides become evident indicating that puromycin released them immediately upon incorporation even in the presence of elongation inhibitors.Essential revisions:The reviewers have different suggestions-one feels that the literature needs to be more fully referenced. One other suggests consideration of the statistical basis for the observations in Figure 2 (has it been repeated?) and that the use of cycloheximide would have made the manuscript more robust. Acceptance of the manuscript does not require additional experimentation however, but include your response to these comments in a revised manuscript.Reviewer #1:The authors of this, and the co-submitted manuscript investigated the assumption that puromycin is a spatial indicator of translating ribosomes. This assumption is based on the puromycylation of nascent chains remaining associated with the ribosome on the mRNA, in effect freezing in its position there in the presence of elongation inhibitors. When detected by antibodies to the puromycin derivative, the signal supposedly indicated the sites of translation.The work presented here convincingly shows that the puromycylated signal comes from released, truncated polypeptides, and not the nascent chains associated with the ribosome. In a straightforward experiment, using the suntag system, the puromycylated nascent chains were not present on ribosomes, but only after the peptides were released from the ribosome did the fluorescence of the tagged puromycylated peptides become evident indicating that puromycin released them immediately upon incorporation even in the presence of elongation inhibitors.The work is of significance particularly since a number of high impact publications have relied on the puromycylation signal indicating the location of ongoing translation. Conclusions that derive from that assumption have to be re-evaluated, since some of them are clearly wrong (e.g. translation in the nucleus) or misleading (work on neurons). I feel it is important to publish this work before more erroneous conclusions contaminate the literature.Reviewer #2:This study by Enam and colleagues re-exams a frequently used assay based on puromycin incorporation to determine the sub-cellular site of mRNA translation. They convincingly show that puromycin treatment results in rapid release of nascent polypeptides, and that this is not inhibited by the translation elongation inhibitor emetine. I agree with the authors that it is important to point out this flaw in the puromycin incorporation assay, and their study does an excellent job in doing this. The main issue here is that there is already data in the literature that shows that nascent chains rapidly (tens of seconds) dissociate from the ribosome and move away from the site of translation, even in the presence of a translation elongation inhibitor (CHX). One example that comes to mind is the paper from the Zhuang lab (PMID: 27153499), where they show that nascent chains are fully released in 1-2 min upon addition of puro and CHX . That being said, the Zhuang lab paper does not make a point out of this result and how it affects interpretation of OPP assays, and I believe that is still useful.Reviewer #3:C. elegans cells and, 3) Lack of co-localization of the SunTag translation reporter with its encoded mRNA in mammalian cells. Authors warn on the use of puromycylation assays as the sole approach to localize translation in cells.Green's group questions the accuracy of the ribopuromycylation method (RPM) to report on the subcellular localization of translation. In RPM, translation elongation inhibitors (emetine and cyclohexamide) preclude the release of puromycylated peptides from the ribosome. Using three approaches, they demonstrate that emetine does not prevent the release of nascent peptides, which diffuse from the ribosome after puromycin treatment: 1) in vitro translation assay of luciferase reporters, 2) Localization of O-propargyl-puromycin (OPP) elongating peptides in the nucleus of Findings from this work will certainly help the scientific community to interpret the data from puromycylation experiments and to choose the best experimental approaches to assess the spatial regulation of translation. The authors state that experiments have been performed once or twice. Hence, the existing experimental evidence could benefit from additional data and analysis to support the main conclusion .1) Only emetine was used to inhibit translation elongation. Adding cyclohexamide to some or all the experiments will validate their conclusions.2) Experiment in Figure 2 has been done only once. The same experiment should be repeated two more times in worms.3) In Figure 3, the media of two experiments is plotted, but the standard deviation is not indicated. The same experiment should be repeated one more time. The experiment in Figure 3—figure supplement 1B has done only once, but it is presented as a major conclusion in the Results section of the paper.4) Figure 4. The number of cells considered for the quantifications is low for a fixed experiment (two experiments of ten cells each). The experiment should be repeated one more time, and authors are encouraged to increase the number of cells they quantify.5) Figure 5 is a simple calculation of diffusion to support the idea that puromycylated peptides can be detected far from their site of translation. Two suggestions to support conclusions from this part of the paper: 1) Improve the mathematical modeling to fit translation localization results obtained from puromycylated experiments in neurons and cell lines, and 2) Support the conclusions from the modeling with experimental data. The latest can be done using their SunTag reporter system in U2OS cells or neurons to track single peptides. Essential revisions:The reviewers have different suggestions-one feels that the literature needs to be more fully referenced. One other suggests consideration of the statistical basis for the observations in Figure 2 (has it been repeated?) and that the use of cycloheximide would have made the manuscript more robust. Acceptance of the manuscript does not require additional experimentation however, but include your response to these comments in a revised manuscript.In response to reviewer comments, we have clarified a number of statements and references in our Introduction and Discussion to provide broader context and clarify methods. We have updated Figure 3 to clarify the range of data obtained from replicate experiments and added data from an additional replicate to Figure 2.In addition, the example untreated cell in Figure 4B was replaced with a more representative image, since the previous image had more mRNAs (~40) than was allowed in our analysis (35), thus making it unrepresentative of the cells that were analyzed. We have also increased the size of the zoom panels in this figure and normalized the red channel to the same values to increase clarity.Reviewer #2:This study by Enam and colleagues re-exams a frequently used assay based on puromycin incorporation to determine the sub-cellular site of mRNA translation. They convincingly show that puromycin treatment results in rapid release of nascent polypeptides, and that this is not inhibited by the translation elongation inhibitor emetine. I agree with the authors that it is important to point out this flaw in the puromycin incorporation assay, and their study does an excellent job in doing this. The main issue here is that there is already data in the literature that shows that nascent chains rapidly (tens of seconds) dissociate from the ribosome and move away from the site of translation, even in the presence of a translation elongation inhibitor (CHX). One example that comes to mind is the paper from the Zhuang lab (PMID: 27153499), where they show that nascent chains are fully released in 1-2 min upon addition of puro and CHX . That being said, the Zhuang lab paper does not make a point out of this result and how it affects interpretation of OPP assays, and I believe that is still useful.We thank the reviewer for putting our study in the broader context of other work in the field. We agree that evidence for release of nascent chains after puromycin addition in the presence of cycloheximide or emetine inhibitors has existed for decades. To our knowledge, this was first shown by Colombo in 1965 and Grollman in 1968; in particular, the Grollman paper showed that there was at most very modest stabilization of nascent peptide on elongating ribosomes in the presence of emetine, while Colombo demonstrated that cycloheximide only transiently stabilized nascent peptides on the ribosome. We also appreciate that the group of single molecule imaging papers (looking at translating ribosomes) emerging several years ago used puromycin release of polypeptides as an experiment to identify elongating ribosomes in real time . The paper from the Zhuang lab referenced by the reviewer  indeed shows a short delay in release of nascent peptide by puromycin in the presence of CHX, but it is not clear if this (as well as the effect seen by Colombo in 1965) is due to stabilization of puromycylated peptides on the ribosome, or slowed reactivity of puromycin with the unusual ribosome state induced by CHX . Regardless of the mechanism, this slight stabilization would still ultimately result in complete dissociation of nascent peptide on the time scales used in puromycylation imaging experiments. Despite this literature, there remains widespread usage of “puromycylation” to localize elongating ribosomes, prompting us to make a specific point of this matter. In particular, given that the work by Yewdell and colleagues made a strong and specific point about the role of emetine in stabilizing puromycylated peptides on ribosomes, we felt it important to specifically address this point. We have added several sentences in the Discussion to more fully acknowledge the literature in this area.Reviewer #3:[…] 1) Only emetine was used to inhibit translation elongation. Adding cyclohexamide to some or all the experiments will validate their conclusions.We chose to perform our experiments with emetine due to its much slower off-rate and the more common use of this inhibitor in studies utilizing RPM. Indeed, in the initial development of this approach, it was argued that emetine was more useful for stabilizing nascent peptides on ribosomes for this reason , though some recent studies utilizing RPM did not include emetine. We expect that CHX would produce similar results as seen in previous studies using metabolic labeling (Colombo 1965) and SunTag live imaging .2) Experiment in Figure 2 has been done only once. The same experiment should be repeated two more times in worms.We have repeated this experiment again to validate our initial observations.3) In Figure 3, the media of two experiments is plotted, but the standard deviation is not indicated. The same experiment should be repeated one more time. The experiment in Figure 3—figure supplement 1B has done only once, but it is presented as a major conclusion in the Results section of the paper.We have updated the graphs in Figure 3 and Figure 3—figure supplement 1B with shading to indicate the full range of values obtained in the replicates. We also clarify that the experiment in Figure 3—figure supplement 1B was performed twice as noted in the figure legend. While we appreciate the fact that repeating an experiment in an identical manner makes the result of a single experiment more robust, the RRL experiments reported in Figure 3B, C and Figure 3—figure supplement 1B are fundamentally similar and were performed independently, and all support the same conclusion that emetine does not prevent release of puromycylated peptides from the ribosome. In light of current limitations on experimental time, we argue that sufficient robustness derives from these similar experiments.4) Figure 4. The number of cells considered for the quantifications is low for a fixed experiment (two experiments of ten cells each). The experiment should be repeated one more time, and authors are encouraged to increase the number of cells they quantify.5) Figure 5 is a simple calculation of diffusion to support the idea that puromycylated peptides can be detected far from their site of translation. Two suggestions to support conclusions from this part of the paper: 1) Improve the mathematical modeling to fit translation localization results obtained from puromycylated experiments in neurons and cell lines, and 2) Support the conclusions from the modeling with experimental data. The latest can be done using their SunTag reporter system in U2OS cells or neurons to track single peptides.The data we obtained from our several fixed imaging experiments are highly consistent with one another and we feel provide sufficient statistical significance to conclude that puromycylated peptides are not retained on the elongating ribosomes, with or without emetine.To address the second point, our modeling parameters are in fact derived from measured diffusion constants for GFP, so we don’t think there is much value in making these measurements for our SunTag peptides. This modeling exercise was simply meant to provide some sense of the time scales for diffusion that would be relevant to experiments routinely reported in the literature.


Advances made in pediatric cardiology, cardiac surgery and critical care have significantly improved the survival rate of patients with congenital heart disease (CHD) leading to an increase in children with CHD presenting for noncardiac surgical procedures. This study aims (1) to describe the trend and perioperative mortality rates in patients with CHD undergoing noncardiac surgical procedures at children’s hospitals over the past 5 years and (2) to describe the patient characteristics and the most common type of surgical procedures. The Pediatric Health Information System (PHIS) is an administrative database that contains inpatient, observation, and outpatient surgical data from 52 freestanding children’s hospitals. Thirty-nine of the 52 hospitals submitted data on all types of patient encounters for the duration of the study from 2015 to 2019. The total numbers of non-cardiac surgical encounters among patients with history of a CHD diagnosis significantly increased each year from 38,272 in 2015 to 45,993 in 2019 (P < 0.001). Despite the increase in case numbers, there has been a significant decline in mortality rates to the most recent incidence of 1.06% in 2019. Careful patient selection and medical optimization of patients aligned with specific expertise at dedicated children’s hospitals may lead to improvement in mortality rate. Future studies comparing the outcomes of patients with cardiac disease based on hospital type and volume as well as type of providers may help determine the future of care including potential need for regionalization of noncardiac care for this vulnerable patient population. Advances made in pediatric cardiology, cardiac surgery and critical care have significantly improved the survival rate of patients with CHD leading to increases in the populations of children and adults with significant CHD3. Children with CHD frequently undergo diagnostic and interventional procedures that require sedation or general anesthesia. Analysis of the Pediatric Health Information System (PHIS) database between 2004 and 2012 demonstrated that 41% of children who had undergone surgery to correct CHD in the first year of life also underwent at least one non-cardiac surgical procedure by age 54. Patients with CHD are generally considered at higher risk for cardiac arrest, perioperative mortality and major morbidity than those without CHD8.The prevalence of moderate to severe congenital heart disease (CHD) in the United States is estimated to be 6 per 1000 live-born full term infants5. From 1994 to 2005, the POCA Registry collected data on 373 anesthesia-related cardiac arrests (CA) in children6. Thirty-four percent of patients (n = 127) had heart disease (HD) . Mortality was 33% in patients with HD as compared to 23% in those without HD6. In a recent study, using the American College of Surgeons National Surgical Quality Improvement Program (ACS NSQIP) database, and after exclusion of the patient without CHD and those with minor CHD, the incidence of mortality was 4.7% in a derivation cohort and 4.0% in the validation cohort9. These investigations were conducted over a period of two decades and utilized different datasets. In addition, none of those studies have looked at changes in number of cardiac patients with CHD requiring noncardiac procedures over time, neither at the change in mortality rate. Therefore, drawing any inferences about trends in mortality of patients with CHD over time is not possible.In a comprehensive analysis of data from the University Hospital Consortium, 191,261 inpatient, non-cardiac procedures occurring between 1993 and 1996 in pediatric patients were analyzed. Mortality was 3.8% in the patients without CHD and 6.0% in the patients with CHD This study aims (1) to describe the trend and perioperative mortality rates in patients with CHD undergoing noncardiac surgical procedures at children’s hospitals over the past 5 years and (2) to describe the patient characteristics and the most common type of surgical procedures performed during that same time interval.10. Only hospitals that submitted data from all encounter types during the entire time period of study were included.The Pediatric Health Information System (PHIS) is an administrative database that contains inpatient , observation (< 24 h admission), and ambulatory (outpatient) surgery discharge/encounter data from 52 freestanding children’s hospitals that are part of the Children’s Hospital Association. The PHIS contains International Classification of Diseases 9th and 10th Revision, Clinical Modification (ICD-9-CM) and (ICD-10-CM) diagnostic and procedure codes, date-stamped billing data, and other administrative data11. Cardiac surgeries and catheter-based interventions were excluded. The frequencies of all noncardiac surgical procedures performed were examined. A non-cardiac surgical procedure was defined as a procedure performed in an operating room (operating room flag in the PHIS database) and with a documented surgical procedure code. The intrinsic surgical risk is the risk of 30-day mortality associated with specific surgical procedures as defined by current procedural terminology (CPT)12. Surgical procedures with CPT risk quartiles 1 and 2 comprise the low-risk procedure category, and quartiles 3 and 4 comprise the high-risk procedures.Patients less than 18 years of age and undergoing a noncardiac procedure between January 2015 and December 2019 who had an ICD-9-CM diagnosis code for CHD (745.0–747.49) or ICD-10-CM diagnosis code (Q 20.0–Q 26.9) at anytime during the past 10 years 2000 to 2019. Patients who did not have an ICD-9-CM or ICD-10-CM diagnosis of CHD were excludedDemographics and patient characteristics are described for encounters of CHD patients undergoing non-cardiac surgery using frequencies and percentages for categorical data, and medians with interquartile ranges (IQR) for continuous data, stratified by year. Mortality rates over time in the entire group and stratified by patient type are expressed as percentages and trends were evaluated using the Cochran–Armitage test across the study years. Trends of the number of cases across the study years were evaluated using generalized linear modeling. The most common cardiac diagnoses based on ICD diagnosis coding is presented by year, as well as the most common non-cardiac procedures with intrinsic surgical risk quartile (ISR). The number of cases based on region of the US is graphically depicted using a heat-map. A two-tailed P value threshold of P < 0.05 was implemented for determining statistical significance. Stata  was used for all statistical analyses.Thirty-nine of the 52 participating hospitals in PHIS submitted data on inpatients, outpatients and observation patient types for the duration of the study from 2015 to 2019. For each year from 2015 to 2019, the total numbers of non-cardiac surgical encounters among patients with history of a CHD diagnosis were 38,272, 40,313, 41,454, 43,854, and 45,993, respectively (P < 0.001). Demographics and patient characteristics of patients with CHD presenting for a non-cardiac procedure from 2015 to 2019 are presented in Table Table The number of encounters of patients without a diagnosis of CHD undergoing non-cardiac surgery increased over time , however the mortality rate in this group did not change significantly across these years .Table The number of noncardiac procedures in patients with CHD at PHIS participating children’s hospitals has increased steadily over the past 5 years. Despite the increase in case numbers, there has been a significant decline in mortality rates to the most recent incidence of 1.06% in 2019.13. This mortality rate is higher than the rate reported in this study. This is most certainly due to differences in the underlying severity of CHD in the two cohorts. In the ACS NSQIP cohort 46.9% of patients had major or severe CHD and 67.9% of patients had unrepaired lesions; in the current cohort > 90% of patients would be classified as having minor CHD. Previous work by our group has demonstrated that in children with CHD, patient comorbidities, and severity of the cardiac lesion are the predominant predictors of 30-day mortality13. These differences in patient demographics as regards CHD severity are likely due to the fact that while the PHIS data includes inpatient, outpatient and observation unit encounters at dedicated children’s hospitals, the ACS NSQIP data includes only inpatient pediatric cases and the participating hospitals include freestanding hospitals, children’s hospitals within larger hospitals, specialty children’s hospitals or general acute care hospitals with a pediatric wing14.In a recent study using the ACS NSQIP pediatric database, children with CHD undergoing noncardiac surgery had a 30-day mortality incidence of 1.7% in the derivation cohort and 1.5% in the validation cohort 15. Among the 13,530 cases included in a subsequent external validation cohort, the incidence of 30-day mortality was 0.21% 16. In a more recent study using the ACS NSQIP database, the overall 30-day mortality in 367,065 surgical cases encompassing 659 unique Current Procedural Terminology codes was 0.34%13. Obviously, these studies speak to the high quality of perioperative care currently rendered to children undergoing surgery.While the mortality rate in CHD patients improved, the mortality rate in patients without CHD remained relatively constant at 0.12% over the 5-year study period. The mortality rate in this cohort is comparable to that seen in other studies. Among the 183,423 children included in the 2012 to 2014 ACS NSQIP database, the incidence of mortality was 0.5%  in the derivation cohort and 0.4%  in the validation cohort4. Assignment of our intrinsic surgical risk (ISR) classification to the procedures performed on patients in this cohort demonstrated that most of the noncardiac procedures performed in the outpatient and observation settings are low risk with an ISR of 113. A limitation of the ISR is that tonsillectomies are not included in the classification as this procedure is not reported to the ACS-NSQIP database from which the ISR was derived. The ISR for procedures performed on the inpatient cohort ranged from 1 to 4. Clearly, the more complex surgical procedures are performed in the inpatient setting as would be expected.The most common types of noncardiac procedures performed on CHD patients remained constant over the 5-year study period. Gastrointestinal and otolaryngologic procedures were the most common. This is consistent with a previous study which demonstrated that general surgical and otolaryngologic procedures to be the most common procedures performed in the first 5 years of life in children requiring infant cardiac surgery17. It has been suggested that regionalization of care at specialized hospitals can reduce mortality for congenital heart surgery as well18. Children with CHD, especially those with unrepaired lesions or with a residual lesion burden and compromised cardiovascular status require an individualized approach to anesthetic and surgical care delivered by trained multidisciplinary teams20. Practitioners including cardiologists, intensivists, and primary care physicians who care for children with CHD must address a number of challenges including identifying the best location for procedures , identifying qualified team , and utilizing the expertise of the non-cardiac subspecialties .Careful patient selection and medical optimization of patients aligned with specific expertise at dedicated children’s hospitals may lead to additional improvement in mortality rate over time in patients with CHD undergoing non-cardiac surgery. It has been demonstrated that collaboration and transparency between institutions leads to reductions in-hospital mortality, duration of mechanical ventilation, length of stay and major complications following pediatric cardiac surgery. In children’s hospitals participating in the Pediatric Cardiac Critical Care Consortium (PC4) outcomes improved over a 2-year period while non-participating hospitals showed no improvement in outcomes over this time interval23. Since the results are based on only the 39 children’s hospitals that participated during the past 5 years by contributing all types of patients encounters, the generalizability of the results is limited. While using ICD 9 and 10 in the PHIS database provides the information about the cardiac diagnosis and surgical procedure, it lacks the granularity in regard to the severity of the cardiac disease which is necessary to further categorize the cardiac disease based on functional severity and degree of residual cardiac lesions. In addition, we looked at the trends over 5 years, which led to the inclusion of ICD 9 in the 2015 data. The ICD 9 coding were converted to ICD 10 in 2015. We used the conversion of ICD 9 to ICD 10 to sum the number of procedures and cardiac diagnosis.This study has several limitations. The PHIS database is an administrative database and miscoding of CHD may occurIn conclusion, this analysis of the PHIS database points out that the mortality rate of patients with CHD undergoing noncardiac procedures is improving despite the increase in number of noncardiac procedures performed per year. Future studies comparing the outcomes of patients with cardiac disease based on hospital type and volume as well as the provider experience  may help determine the future of care including potential need for regionalization of noncardiac care for this vulnerable patient population.


The Sessions Planning Committee and Chair should be congratulated for their work on the 2017 Heart Rhythm Society Annual Scientific Sessions held in Chicago, IL, as it was another phenomenal meeting. This year was an example of my favorite saying, “As so goes atrial fibrillation (AF), so goes electrophysiology”; namely, there were many fascinating aspects of AF presented during the late-breaking clinical trials, and there seems to be a shift occurring from interest in primary ablation to interest in the prevention of thromboembolism. Here are a few highlights from this year’s show.1 the study’s safety data at the time of initial review met criteria to continue the trial. There was also an elegant live case streamed from Northwestern University of combined cryoballoon  AF ablation and LARIAT™  LAA ligation.The left atrial appendage (LAA) has quite possibly dethroned rotors as the hot topic in AF, both for cerebrovascular accident (CVA) prevention and in catheter ablation. SentreHEART  announced that, regarding the Left Atrial Appendage Ligation with the LARIAT™ Suture Delivery System as Adjunctive Therapy to Pulmonary Vein Isolation for Persistent or Longstanding Persistent Atrial Fibrillation (aMAZE) trial,2 on patients receiving the WATCHMAN™ device . The initial randomized controlled trials (RCTs) for the device involved patients who were candidates for oral anticoagulant (OAC) therapy, and the initial pivotal trials required that OAC therapy be administered for a minimum of 45 days following initial device implantation.4 The EWOLUTION trial followed more than 1,000 patients after initial implantation as part of a multicenter registry. The population in the EWOLUTION trial was a high-risk population as compared with that seen in prior RCTs with regards to both stroke and bleeding, with an average CHA2DS2-VASc score of 4.5 and with 73% of patients having some contraindication to OAC therapy. Fifty percent of patients had a CHA2DS2-VASc score of ≥ 5 and 40% had a HAS-BLED score of ≥ 4.Dr. Lucas Boersma of St. Antonius Hospital in the Netherlands presented the results of the EWOLUTION trialConsistent with the increasing experience with the device, the researchers running the trial demonstrated a lower risk of serious adverse events than what was seen in either the PROTECT or PREVAIL trials, and a high implant success rate of 98.5%. Only 27% of patients were treated with OAC post implantation, with 60% being given dual antiplatelet therapy. The researchers also demonstrated a marked reduction in CVA and bleeding compared with projected rates. They concluded that the WATCHMAN™ device can safely be used in patients who have a contraindication to OAC therapy, and demonstrated dramatic efficacy despite the majority of patients not undergoing OAC therapy post implantation. More importantly, this data paves the way for a prospective study evaluating whether or not OAC therapy can be completely avoided in patients receiving this type of therapy.5 a prospective, single-arm open-label trial involving patients receiving a REVEAL®  implantable loop recorder who were at high risk for CVA and who had no prior history of AF. Fifty percent of the patients were implanted because of complaints of palpitations. The mean CHA2DS2-VASc score was 4.4. The primary endpoint was AF lasting more than six minutes at 18 months. The decision to use six minutes as an endpoint is based on the previously published ASSERT trial,6 which investigated the significance of AF detected with the use of cardiovascular implantable electronic device monitoring. In the REVEAL-AF study, 29.3% of patients were seen to have AF at 18 months, and 40% by 30 months. The authors concluded that three-quarters of patients would have gone undiagnosed if monitoring had only been continued for 30 days.Dr. James Reiffel of Columbia University presented the primary results of the REVEAL-AF study,7 The REVEAL-AF study now considers this same concept applied to high-risk patients without a history of CVA. The authors also report that 56.3% of patients were started on OAC therapy based on the diagnosis of AF (non-protocol driven).Given the tremendous morbidity of AF-associated CVA, aggressive monitoring for AF in high-risk patients is a good concept. The CRYSTAL-AF study previously demonstrated a high incidence of AF in patients with cryptogenic stroke.8 Interestingly, there was a small but notable decrease in the risk of dementia when antiplatelet therapy was initiated within 30 days . However, there was a profound benefit when OAC therapy with warfarin was started within 30 days of diagnosis, with no demonstrable incidence of dementia, compared with a 0.4% incidence when warfarin was started later. They demonstrate a clear temporal relationship between timing of OAC or antiplatelet therapy initiation and lifetime dementia risk.The group at Intermountain Health in Utah has given us further insight into the timing of OAC therapy for AF. Having previously demonstrated the relationship of AF and microemboli and clinical dementia, Dr. Jared Bunch reported data investigating the relationship of dementia risk to the timing of OAC or antiplatelet therapy once a diagnosis of AF was made. In a patient cohort of over 26,000 patients, in whom almost 22,000 were started on antiplatelet therapy and 4,400 on OAC therapy, Bunch and colleagues compared the incidence of dementia in patients who started OAC therapy within 30 days of AF diagnosis, or between 30 days and one year after AF diagnosis.9 presented during the late breaking clinical trials introduced listeners to the concept of “tailored anticoagulation,” which takes advantage of the rapid onset of non-vitamin-K oral anticoagulants (NOACs). This was a prospective, randomized controlled trial in patients with implanted CIEDs with a primary endpoint of days on OAC therapy. This trial, sponsored by Abbott Medical  enrolled 61 patients with CIEDs equipped with remote monitoring and who were on stable NOAC therapy. Forty-eight of the 61 were randomized to the tailored therapy arm. Patients were monitored biweekly and were allowed to stop OAC therapy if no AF was seen for 30 days. Data collected indicated the use of NOACs decreased by 75% in the treatment arm. There was no incidence of CVA seen in the trial. Further investigation is clearly warranted, but this approach could certainly improve patient compliance and decrease cost and bleeding risk as compared with continuous therapy.These powerful data demonstrate the importance of not just appropriate OAC initiation but also the necessity for rapid initiation and improved patient compliance. One such method could be intermittent OAC use associated with a prompt diagnosis. The TACTIC-AF study10 Dr. Sanchez and his colleagues at the University of California San Francisco and Cardiogram  presented some fascinating data on the use of a smartwatch to diagnose AF. The mRhythm trial investigates the ability of a smartwatch to detect AF with use of its built-in photoplenthysmography sensors.11 The application goes through a “learning phase,” employing deep neural networks to predict patterns in an individual’s heart rate, and then a test phase in-person when the subjects presented for cardioversion using a Kardia smartphone ECG . There were approximately 6,400 subjects, of which there were 440 who had AF and were evaluated as part of a necessary phase for the system to “learn,” and 50 in the test phase who presented for cardioversion. The application demonstrated 98% sensitivity and 90% specificity in diagnosing AF. These tools that are in the realm of “body computing” can lead to treatments that are both more effective and patient-centric, and visionaries such as my partner Dr. Leslie Saxon at the University of Southern California Keck School of Medicine would predict that this is only the beginning.The use of smartwatches by the general population is rapidly increasing and becoming as ubiquitous as smartphones . Smartphones are already being used to diagnose AF.So, based on the 2017 Annual Scientific Sessions, our patients with AF or those high-risk patients without can diagnose themselves with AF using their smartwatch and take their NOAC accordingly, and by doing so, prevent dementia. If they can’t take OAC, they can have LAA exclusion with simply antiplatelet therapy.12 It is certainly true that most people seek the definition of ablation success that is reported in the literature—that is, when the patient is arrhythmia-free beyond the blanking period off antiarrhythmic drugs (AADs). This is arbitrary considering that a previously ineffective medication may become effective after a catheter ablation. The POWDER AF trial randomized patients undergoing their first contact force-guided ablation procedure who were free of arrhythmia during the three-month blanking period to either continuation or discontinuation of their previously ineffective AADs. At 12 months post ablation, a significantly higher percentage of patients remained arrhythmia-free as compared with those who stopped their AADs at three months . Not surprisingly, patients had fewer unscheduled visits and improved symptoms. The authors conclude that combined therapy after a first ablation may result in improved outcomes.Abstracts on catheter ablation for AF once again were a large part of the 2017 Annual Scientific Sessions, but I do not think that I’m unique in my opinion that we are in a period of refinement of this procedure  while we wait for the “next big thing.” Not to completely leave out catheter ablation, the results of the POWDER AF trial were presented at the late breaking clinical trials by Dr. Mattias Duytschaever.13While not surprising, the results of this study raise an important issue regarding clinical practice. We typically follow prior clinical trial design, which is designated primarily to demonstrate a benefit or superiority of a therapy. A clinical benefit such as that demonstrated in the POWDER AF trial should not be discounted. In addition, it will be interesting to see if there is a long-term benefit if AAD use is discontinued. This would be reminiscent of data originally presented at the Annual Sessions in which the AF-free period after epicardial fat pad botulinum toxin injection in patients undergoing open heart surgery long outlasted the autonomic effects.AF has been a robust subject at many of the Heart Rhythm Sessions, and the 2017 Scientific Sessions were no exception. I hope these “highlights,” as subjective as they may be, spark a continuing interest in AF investigation, but more importantly, that they translate into improved care for our patients.MD, FHRSRahul N. Doshi, rahuldoshimd@me.comAssociate Professor of Clinical MedicineDirector of Cardiac ElectrophysiologyKeck School of MedicineUniversity of Southern CaliforniaLos Angeles, CA 90033


We developed an evaluation method combining longitudinal imaging analysis in vivo with simultaneous manipulation on single cell level. An abdominal imaging window was implanted in vivo in Balb/C mice for recurrent imaging after implantation. Intravenous injection of Fluorescein Isothiocyanate (FITC)-Dextran was used for labelling of vessels and Rhodamine 6G for hepatocytes. Minimal cell injury was induced via ablation with a femtosecond laser system during simultaneous visualisation of targeted cells using multiphoton microscopy. High-resolution imaging in vivo on single cell level including re-localisation of ablated regions in follow-up measurements after 2–7 days was feasible. Targeted single cell manipulation using femtosecond laser pulses at peak intensities of 3–6.6 μJ led to enhancement of FITC-Dextran in the surrounding tissue. These reactions reached their maxima 5–15 minutes after ablation and were no longer detectable after 24 hours. The procedures were well tolerated by all animals. Multiphoton microscopy in vivo, combined with a femtosecond laser system for single cell manipulation provides a refined procedure for longitudinal evaluation of liver micro-regeneration in the same region of interest. Immediate reactions after cell ablation and tissue regeneration can be analysed.The liver is known to possess extensive regenerative capabilities, the processes and pathways of which are not fully understood. A necessary step towards a better understanding involves the analysis of regeneration on the microscopic level in the  The liver not only fulfils a broad spectrum of functions in the human body: from the metabolism of toxins to the synthesis of proteins and clotting factors to the storage of energy via glycogen. Several studies could also demonstrate, that the liver possesses high regenerative capacities: In the rodent model, loss of up to two thirds of the liver mass could be compensated within a week after partial hepatectomy –3. This Against this background arises the need to further analyse the processes involved in liver regeneration, for example, on the cellular level to reveal the types of cells responsible for recovery of damaged regions. Ideally, the examination method would leave not only the imaged tissue unharmed, but would also allow the examined animal to stay alive for longitudinal analysis at further time points. Most standard protocols for intravital imaging of the liver involve the animal’s death after imaging , 9. CellRitsma et al. described already in 2012 –15, thisMultiphoton microscopy (MPM)  is similin vivo in the liver. In earlier studies, UV laser beams were used to ablate cells in abdominal tissue, for instance, in the intestine [11−1013 W/cm2 at pulse durations of 10–100 fs are needed to achieve this effect: Seed electrons are generated in the focal volume, which are accelerated and can lead to the formation of a plasma in the focal volume. This can induce precise, abrupt, and complete destruction of the tissue through the formation of a cavitation bubble [With this project, we are the first to combine high-resolution imaging with a method for targeted cell manipulation on a micrometre scale ntestine . Howevern bubble , 20. Duen bubble  and is tTo reach our objective of establishing an optimised imaging technique for longitudinal analysis and follow-up examination of targeted tissue damage, we combined the intravital multiphoton microscopy and the implantation of an abdominal imaging window with a femtosecond amplifier ablation laser system for microscopic tissue manipulation see . This prFor the imaging setup, a Chameleon Ultra II  laser system with a repetition rate of 80 MHz, tuneable for wavelengths from 680 to 1080 nm was used. The best possible imaging wavelength was determined at the beginning of measurements in an analysis with the chromophores used for staining as 780 nm. The beam was then led through an attenuator complex consisting of a λ/2-plate and a polarising beam splitter (PBS) to the first beam expansion unit . A modified Thorlabs MPM200 microscope  was used for multiphoton microscopy. Inside the microscope, the imaging beam was combined with the manipulation beam through another PBS.The manipulation beam was produced using a Spitfire Solstice Ace laser system  running at a central wavelength of 800 nm and a repetition rate of 100 Hz. The beam was led through an attenuator complex (λ/2-plate and PBS) and then through a mechanical shutter allowing for the generation of single pulses. An oscilloscope was used for control of emitted pulses. After passing through a second λ/2-plate for fine tuning of the applied laser power, the manipulation beam was coupled into the microscope’s PBS. The ablation laser power at the back aperture of objective was approximately 31–66 mW resulting in pulse energies of 3–6.6 μJ. Together, both beams passed through the microscope’s dichroic mirror, the 20x objective and into the tissue or probe. Here, the imaging beam excited the tissue’s molecules to emit light which is reflected back into the microscope and passes via the dichroic mirror to the two bandpass filters and photomultiplier tubes (PMT). The signals of the two PMTs were recorded and analysed using the imaging software ThorImageLS 1.4.Data was acquired using ThorImageLS 1.4 as z-stacks, with 0.02 mm layer thickness, resulting in stacks about 1–2 mm thick, or as time-stacks, with one image every 0.1 seconds, resulting in stacks of up to 50 images. Data analysis was performed using the open platform software ImageJ  for the in vitro testing of imaging and ablation before moving to resected organs and in vivo experiments, cultures of the canine pleomorphic mammary adenoma cell line ZMTH3 were used. Cells were cultured in petri dishes in supplemented RPMI medium with the selection antibiotic G418 and split when necessary . Cells expressed mitoDsRed, a red fluorescent protein (RFP), which is tagged to mitochondria, and were stained with Calcein AM .For Implantation of an abdominal imaging window was performed in 20 BALB/c mice as described by Ritsma et al. . The abdTo achieve a comprehensive and as complete postoperative assessment of the animals as possible, the scoring system mentioned in The study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Lower Saxony State Office for Consumer Protection and Food Safety (LAVES) under the number 17/2715. All surgery was performed under isoflurane anaesthesia, and all efforts were made to minimize suffering.All animals were imaged on days 7, 8, 10 and 14 after implantation using a multiphoton microscope. An immersion objective was used with Vidisic eye gel as immersive agent. Again, induction and maintenance of anaesthesia were secured by inhalation of isoflurane. For visualisation of blood vessels, 10 mg/ml fluorescent FITC-Dextran solution combined in 100 μl of saline with 1 mM Rhodamine 6G  for staining of hepatocytes was injected into the warmed tail vein. During imaging, the mouse was placed on a warming mat with rectal control of body temperature and eyes were covered with bepanthen or similar dexpanthenol-containing balm. Microscopy was performed to a maximal duration of 4 hours. Every hour, an injection of 100 μl of 20% glucose-solution was given intravenously.For the analysis of the regenerative capacities of the liver, the first imaging session was used for targeted cell manipulation in the sense of single-cell ablation. This precision is made possible by use of the above-mentioned femtosecond laser system which allows defined laser application without affecting neighbouring cells. The required laser power was determined beforehand (data shown in paragraph 4.3.2). Post-ablation imaging was continued for an hour, bevor the animals were woke up. The targeted region was marked on the titan ring to facilitate re-localisation in the follow-up imaging 1, 3 and 7 days after ablation. At the end of the last imaging session, the animal was euthanized via cardiac puncture in accordance to animal welfare regulations under continued isoflurane anaesthesia and organs resected for further analysis.All in all, the animals well tolerated not only the implantation of the AIW, but also the subsequent ablation and imaging sessions. Twenty mice were included in the study, two of which died peri-operatively without detectable causes. All other animals tolerated narcosis and implantation without any complications and hardly any weight loss. Further scoring was based on the animals’ general appearance and behaviour  sufficed for the staining to arrive in the liver. Ablated regions could be re-localised using the same targeting system as mentioned above for imaging.In preceding measurements, the imaging parameters were analysed. For imaging, a wavelength of 780 nm was found to deliver the best possible intensities of fluorescence and contrast for our combination of staining with appropriate penetration depth into the liver tissue. For a comparison of fluorescence signals induced by different excitation wavelengths see ex and in vivo–see Figs in vivo imaging as a time stack, the position change of intravascular structures could be observed and consecutively an estimate for the blood flow velocity calculated as 25.65 μm s-1 (2.565 μm in between pictures 0.1 seconds apart).For all imaging sessions, staining worked very well to sufficiently depict cells and cell structures (Rhodamine 6G) as well as tissue architecture in comparison to blood vessels/capillaries (FITC-Dextran). The success was independent of the object of investigation  could be detected directly surrounding the observable microscopic tissue destruction. As FITC-Dextran is typically applied for visualisation of changes in cell permeability, one can presume that in case of manipulation of single cells with the ablation laser, this enhancement is due to damage in the cell membrane. During the further course of imaging in 5 minute intervals after ablation, the area showing increase of FITC-Dextran around the manipulated cells first expanded and then receded again see . The maxIn this study, we aimed to establish a method for targeted single-cell manipulation during longitudinal imaging for follow-up examination of the tissue micro-damage. We combined intravital multiphoton microscopy  through As already outlined above, the mechanisms involved in regeneration of the liver after tissue injury are not yet fully understood. Especially for analysis on a microscopic/cellular level, new techniques like those described in this project can help to gain further insights.-1 is significantly lower compared to blood flow velocities observed by other research groups calculating capillary hemodynamic in the skin with velocities of around 192 μm s-1 [-1 [The applied dyes stained the structures intended for imaging satisfactorily, enabling the differentiation between tissue and vessels for determination of e.g. blood flow velocity. Our estimate of about 25.65 μm s s-1 [-1 . This di s-1 [-1  describeWith multiphoton microscopic imaging through an AIW, longitudinal imaging of all organs and/or tissues accessible from the abdominal (or thoracic) wall, such as liver, bowel, stomach, lungs, etc., is possible. Furthermore, dynamic imaging is possible in the acute and long-term setting: Alterations made to cellular integrity or tissue structures can be observed and recorded simultaneously. By implantation of the abdominal window, a closed system was achieved, which insured the animals’ survival and made longitudinal analysis after ablation possible. We could verify the observations described by Ritsma et al.  for AIW For ablation in ZMTH3 cell culture as well as livers ex vivo (through an AIW), an average of 5 pulses at 0.5 μJ pulse energy sufficed for manipulation of cells, whereas 5 pulses at 6 μJ pulse energy were needed for sufficient ablation in livers in vivo, corresponding to energy 12 times as high. This difference might be caused by the necessary penetration depth and by the surrounding tissue. It is however unlikely, that massive energy transport during the pulse by blood flow or cell transport occurs, as our high pulse energy femtosecond laser pulses induce nonlinear interactions such as multiphoton ionization but do not lead to an accumulation of heat.in vivo, the maximal areal extent of FITC-Dextran enhancement was reached after 5 minutes, reduced by 30% after 20 minutes and was no longer detectable after 40 minutes. This very dynamic process clearly indicated that the tissue was ablated and not only photobleached. For cellular, FITC-positive reactions, the maximum was reached after 15 minutes, indicating a slightly slower reaction time of cellular invasion as compared to instantaneously increased permeability of injured cells. These reactions as well were no longer detectable 40 minutes after ablation. In controls 1, 3 and 7 days after manipulation, no change in tissue structure was observed. These findings imply rapid micro-regenerative reactions, clearing up for example cell debris, leading to the restitution of the tissue architecture when the extent of injury was only on a micrometre scale. These apparently very quick reactions are highly interesting and should be analysed in more detail by continuous imaging in the first minutes after ablation.After ablation Studies have shown liver regeneration upon acute injury of small parts of the liver mass to induce regeneration in form of hypertrophy, mediated mainly by mature, periportal hepatocytes , 28. We 4), mechanic damage  or hypoxia (warm ischemia). Analysis of reactions on these mechanisms would also enable an evaluation of the influence of local (cell level) versus systemic (organ level) injury.The targeting of more detailed ablation in the lobule would potentially allow the analysis of the direction of cell migration in the course of regeneration and a possible involvement of stem- and progenitor cells. Furthermore, another interesting aspect would be the comparison of regenerative mechanisms upon different kinds of tissue injury: Targeted laser-based micro-manipulation versus “conventional” mechanisms such as toxins  Average weight development after implantation of an AIW during the course of experiments. (B) Development of different scores Click here for additional data file.S1 File(XLSX)Click here for additional data file.


Non-pharmacological treatment (NPT) improves cognitive functions and behavioural disturbances in patients with dementia, but the underlying neural mechanisms are unclear. In this observational study, 21 patients with dementia received NPTs for several months. Patients were scanned using magnetoencephalography twice during the NPT period to evaluate NPT effects on resting-state brain activity. Additionally, cognitive functions and behavioural disturbances were measured using the Mini-Mental State Examination (MMSE-J) and a short version of the Dementia Behaviour Disturbance Scale (DBD-13) at the beginning and the end of the NPT period. In contrast to the average DBD-13 score, the average MMSE-J score improved after the NPT period. Magnetoencephalography data revealed a reduced alpha activity in the right temporal lobe and fusiform gyrus, as well as an increased low-gamma activity in the right angular gyrus. DBD-13 score changes were correlated with beta activity in the sensorimotor area. These findings corroborate previous studies confirming NPT effects on brain activity in healthy participants and people at risk of dementia. Our results provide additional evidence that brains of patients with dementia have the capacity for plasticity, which may be responsible for the observed NPT effects. In dementia, NPT might lead to improvements in the quality of life. It is characterised by deterioration in cognitive functions, behaviour, and psychological symptoms. To date, there are only a few pharmacological treatment options available to influence the course of dementia4. At the time a patient is diagnosed with dementia, the brain damage is considered too severe to be reversible to a healthy state5. Thus, early diagnosis and interventions are essential to treat people at risk of dementia, such as those with “cognitive impairment, no dementia” (CIND) or “mild cognitive impairment” (MCI)9. Recent studies showed that MCI can be reversed to normal cognitive functions by non-pharmacological treatments (NPTs)15. Neuroimaging studies revealed that NPTs enhance brain plasticity even in older adults17 supporting the importance of early diagnosis and interventions in dementia.Dementia is a chronic and progressive syndrome caused by brain diseases23. However, the neural mechanisms underlying the positive influences of NPTs remain unclear.The positive effects of NPTs are not limited to people at risk such as those with CIND or MCI. Clinicians and therapists frequently observe that NPTs improve cognitive functions and/or behaviours in patients with dementia21. Although cognitive improvements are the secondary target, patients in these facilities often show progress in cognitive functions, as well as in behavioural and psychological symptoms of dementia (BPSD)20. Based on these experiences, clinicians and therapists are often under the impression that patients with dementia still exhibit sufficient brain plasticity to respond to NPTs; however, evidence for this is sparse in literature. In this study, 21 patients with dementia were recruited at our geriatric health service facility. They received NPTs for several months and their resting-state brain activities were measured using magnetoencephalography (MEG) two times during the total NPT period. The brain activity recordings were compared to better understand how NPT changes the resting-state brain activities in patients with dementia.Geriatric health service facilities are transitional facilities between hospital and home or nursing home where registered physical, occupational, and speech therapists provide NPT and nursing care to reduce the patients’ hospital stay. Patients with dementia are often transferred there when their physical conditions are usually not too severe to be treated in hospitals but their cognitive symptoms prevent them from staying at their home. The primary goal of the facility is to improve the patients’ behavioural functions, to enable them to live again at homep = 0.002, using bootstrap method). The changes in the MMSE-J score did not correlate with the patients’ age, duration of the NPT period, or the initial MMSE-J score. Taken together, the cognitive functions of the study population improved over the NPT period but the patients’ age nor their initial cognitive function was a predictor of this change.The average MMSE-J score was 12.4 ± 6.6 at the beginning and 14.4 ± 7.8 at the end of the NPT period  at the group level. The change in the DBD-13 score did not correlate with age, duration of the NPT, or initial DBD-13 score.The average DBD-13 score changed from 10.6 ± 10.8 at the beginning of the NPT period to 9.9 ± 11.1 during its end. This score decreased  in 12 patients, increased in 4 patients, and remained unchanged in 2 patients. The improvement of the DBD-13 score was not statistically significant  and the end  of the NPT period. However, the changes in MMSE-J and DBD-13 scores showed no statistically significant correlation .The MMSE-J and DBD-13 scores were negatively correlated at the beginning , whereas only the alpha2 intensity exhibited a decline in the right fusiform gyrus (p = 0.028 at the cluster-level) . The results in all other frequency bands did not reveal any significant changes.The source intensities were compared between the two scans separately at each frequency band. The alpha1 and alpha2 intensities decreased at the second scan in the right temporal lobe . No other significant correlations were found between source intensities and the time between the scans or the behavioural scores. These results indicate that patients with reduced beta intensity near the right sensorimotor area showed a decline in behavioural disturbances.Correlations of the MEG source intensities to the interval between the two scans and the changes in behavioural scores (MMSE-J and DBD-13) were also examined. Changes in beta source intensity close to the sensorimotor area of the right hemisphere were positively correlated with changes in DBD-13 scores (To determine whether the NPT induced different effects in patients with vascular dementia (VD) and Alzheimer’s disease (AD), we averaged the source intensity changes for patients with VD and AD separately and compared them visually Fig. . AlthougThe present study revealed two major findings: (1) NPTs improved cognitive functions in patients with dementia, although behavioural disturbance parameters were not significantly modified, and (2) the spontaneous neural oscillations (i.e. the resting-state brain activity) in the right hemisphere changed over the NPT period and parts of these changes correlated with changes in the behavioural score (DBD-13).5. This idea is supported by the fact that patients with CIND and MCI show improvements in their cognitive functions after NPTs23, and neuroimaging studies have revealed that these patients still show signs of brain plasticity17. The present study applied this idea to patients with dementia. We demonstrated that patients with dementia responded to NPT and that the intervention improved the cognitive functions, as reflected by the MMSE-J score. This improvement did not correlate with either age nor initial MMSE-J score, indicating that the potential of NPT is not limited to young patients or patients with mild dementia. A previous study demonstrated that pathological changes are not always in agreement with the clinical symptoms24; patients with less brain damage may exhibit more severe dementia symptoms and vice versa. It is, however, reasonable to assume that some patients with dementia have the potential to improve their functions similar to that of patients with CIND or MCI.It has been suggested that brains of patients with dementia are severely damaged thus negatively influencing brain plasticity. This is considered to be the reason why these patients do not respond to pharmacological treatments25. The effects of NPTs were independent of the pathological changes in the brain during disease state causing dementia, therefore NPTs are effective in patients with different subtypes of dementia such as VD and dementia due to AD. This is consistent with the novel concept of NPT, which focuses on each patient’s function rather than pathophysiological or neurological causes of their disease21. In the present study, we further demonstrated that NPT induced similar changes in the brain activities of patients with either VD or dementia due to AD  neuronal activities29 which is correlated to BPSD30 and ultimately, neural plasticity31. It is plausible that changes in beta intensity in the sensorimotor area reflect the changes in neuroplasticity or neural network efficiency caused by NPTs through the whole brain network system. Another study using functional MRI in healthy older adults revealed that NPT changes brain activities in a widespread network of frontal, parietal, temporal, subcortical, and occipital regions as well as in the thalamus and the caudate10. This is supported by additional studies using functional MRI in patients with MCI showing that NPTs modify brain activities within a large network that includes the frontal, temporal, and parietal areas12. In the present study, the resting-state activity of the alpha band was altered in the right temporal lobe and the right fusiform gyrus. A larger part of the alpha activity is produced by reciprocal connections between the neocortex and the thalamus33. Previous studies indicate that NPTs modulate large neural networks including the thalamus12. The temporal lobe and the fusiform gyrus are anatomically close to the hippocampus and the right hippocampus has been implicated in dementia-related psychosis34. NPT effectively reduces BPSD35, thus it is a reasonable expectation that NPT alters the spontaneous neural oscillation of the alpha band in the right temporal lobe and the fusiform gyrus. A functional MRI study has shown that NPTs induced brain activity changes in the right angular gyrus and the anterior portion of the left lingual gyrus of patients with AD36. Our result showed that gamma intensity in the right angular gyrus increased after NPT. The angular gyrus is one of the brain network cortical hubs37 which influences various cognitive functions. It is located in the parietal cortex where ageing and dementia bring about changes at an early stage (Retorogenesis theory)39. Lesions in the angular gyrus cause symptoms similar to those in BPSD41. Gamma activity is also correlated with the activities of GABAergic neurons42, and a reduction in GABA is associated with poor cognition44. We speculate that changes in gamma at the angler gyrus reflected the change in neural network efficiency induced by NPTs to compensate for the functional deficit.Previous studies have shown that NPTs have beneficial effects on healthy older adultssee Fig. . The pri47 as it can detect synaptic transmission underlying macroscopically observable brain activity48. In dementia research, MEG demonstrated various advantages over MRI. In practice, MRI scans are often difficult and risky for patients with dementia36. For example, patients with dementia unintentionally bring metallic items into the scanner at times, which is highly dangerous due to the strong magnetic field produced by an MRI scanner. It takes up to 10 s to abort a scan and rescue the patient in the case of an emergency. Moreover, patients are often not happy with an MRI scan as the device creates loud noises. The patients also feel nervous in the confined space of the MRI gantry and must keep still inside the scanner without having any company. By contrast, a MEG generates neither a magnetic field nor noise. Patients are not opposed to this technique as they are not physically restricted by the scanner and another person can attend to them during the scan to ensure their safety. A scan is completed within 10 min or less including preparations, can be immediately aborted anytime, and in case of an emergency, the patient can be taken out within 10 s. Additionally, MEG scans provide clinicians with an opportunity to detect epileptic discharges. Of patients with dementia, 10–22% have epilepsy as a secondary diagnosis and it is not easy to diagnose epilepsy in older adults because their seizures are often non-convulsive52. In fact, in the present study, 2 out of the 21 patients presented epileptic discharges during the scan and were excluded from further analyses so that they may be treated with anti-epileptic drugs. In summary, the present study restated that MEG is an ideal neuroimaging technique for dementia studies.In the present study, we used MEG data instead of MRI or functional MRI data. MEG is a non-invasive neuroimaging technique that is sensitive to changes in brain activity related to dementiaThis study has five limitations: (1) The group of patients with dementia was heterogeneous in terms of their pathologies of the brain diseases causing dementia (e.g. stroke or Alzheimer’s diseases), thus making it challenging to fully evaluate how NPTs affected pathological changes of the diseased brain. However, as shown in Fig. Taken together, NPTs can change the resting-state brain activity and improve dementia symptoms. These changes are based on neural plasticity and not limited to patients in pre-dementia conditions (CIND and MCI). Clinicians and therapists should pay more attention to primary care patients with dementia because these patients have the potential for improvement with NPTs.A total of 21 patients with dementia  were enrolled from our geriatric health services facility ‘Kakehashi’ regardless of the brain disease causing dementia (e.g. stroke or AD). The group of patients with dementia was an epitome of the population of dementia in the region. The dementia diagnosis was established by clinicians prior to patients’ admission to the facility. Most patients were affected by AD with other types of dementia also being present in the study population 55. Each patient completed a set of behavioural assessments  two times during NPT period  and a lower score indicates a less problematic behaviour . There is no reference value for the DBD-13.The levels of cognitive impairments and behavioural disturbances in patients were assessed by therapists using the Japanese version of the Mini-Mental State Examination (MMSE-J)ale DBD-155. Each All patients visited our MEG centre at Hokuto Hospital two times during the NPT period Fig. . The 3-mhttps://www.fil.ion.ucl.ac.uk/spm/) and the MEAW system  . No source priors were used for source estimation. The source images were smoothed (20 × 20 × 20 mm) and taken to the second (group)-level analysis.Of the 21 patients, 3 patients were excluded from analysis: 2 exhibited epileptic discharges during the MEG scans and 1 induced severe artefacts due to dyskinesia. The remaining 18 patients were analysed offline using the software package SPM-12  = 0.05 and a cluster extent at k > 10 (=80 mm3)62. Cortical areas at which the peaks of the estimated sources were located were identified using the SPM-12 software.Two types of the second (group)-level analyses were carried out: (1) to find brain regions in which intensities of the spontaneous neural oscillation were different between the two scans, and (2) to find brain regions in which changes in source intensities were correlated with changes in MMSE-J scores, DBD-13 scores, or the time between the two scans. For the first analysis, the source images were compared within each patient using the paired p (uncorrected) = 0.001 and a cluster extent at k > 10.After the main analyses, we performed two additional analyses to determine whether the NPT induced different effects on patients with VD (n = 4) and AD (n = 11). For the first step, the changes in source intensities between the two scans were averaged over patients with the same pathological labelling  using the Image Calculator; the averaged images were visually inspected Fig. . For theBootstrapping approaches were used to evaluate changes in MMSE-J and DBD-13 scores over NPT period considering a relatively small number of patients (n = 18). First, changes in these scores between pre- and post-NPT were evaluated. The difference between the pre- and post-NPT scores was resampled with 10,000 times replacement across the 18 patients, and the percentage of the resampled differences, being larger or smaller than 0 , was taken as the significance level. Second, the correlations between the patients’ age, the duration of the NPT period, and the MMSE-J and DBD-13 scores were evaluated. For each pair of variables, the correlation coefficient was calculated from resampled with replacement data across the 18 patients for 10,000 times, and percentage of the resampled coefficients, being larger or smaller than 0 , was taken as the significance level. Statistical analyses were performed using MATLAB .


ReQoL-10 and ReQoL-20 have been developed for use as outcome measures with individuals aged 16 and over, experiencing mental health difficulties. This paper reports modelling results from the item response theory (IRT) analyses that were used for item reduction.From several stages of preparatory work including focus groups and a previous psychometric survey, a pool of items was developed. After confirming that the ReQoL item pool was sufficiently unidimensional for scoring, IRT model parameters were estimated using Samejima’s Graded Response Model (GRM). All 39 mental health items were evaluated with respect to item fit and differential item function regarding age, gender, ethnicity, and diagnosis. Scales were evaluated regarding overall measurement precision and known-groups validity .The study recruited 4266 participants with a wide range of mental health diagnoses from multiple settings. The IRT parameters demonstrated excellent coverage of the latent construct with the centres of item information functions ranging from − 0.98 to 0.21 and with discrimination slope parameters from 1.4 to 3.6. We identified only two poorly fitting items and no evidence of differential item functioning of concern. Scales showed excellent measurement precision and known-groups validity.The results from the IRT analyses confirm the robust structure properties and internal construct validity of the ReQoL instruments. The strong psychometric evidence generated guided item selection for the final versions of the ReQoL measures.The online version of this article (10.1007/s11136-020-02622-2) contains supplementary material, which is available to authorized users. While there are patient-reported outcome measures (PROMs) focusing on the process of recovery from mental health problems , a revieThe EQ-5D has been adopted in the UK for routine outcome measurement and is preferred by the National Institute for Health and Care Excellence (NICE) to calculate Quality-Adjusted Life Years (QALYs) for use in cost-effectiveness analyses . While iVarious stages of development process have been described in detail –23, therIRT has become the dominant psychometric theory informing the quantitative development of patient-reported outcome measures (PROMs) , 29. OurThe aim of this paper is to describe the use of IRT analyses in Stage 3 of the development process. The objectives of the IRT analyses are to identify any ReQoL items that show poor fit to the IRT model, to calculate the item information functions to identify the score range where each item provides the most information, and to identify any potentially problematic differential item functioning with respect to age, gender, ethnicity, and diagnosis.Participants with a mental health diagnosis were recruited from 20 secondary care organisations (67% of sample), three general practice surgeries (27%), three charity organisations 1%), and a cohort of trial participants without depression who had expressed an interest in being included in future-related research (5%). The sample is discussed in detail elsewhere %, and a . Data conone of the time, only occasionally, sometimes, often, and most or all of the time. Negatively worded items were reversed, so that higher values represent better recovery of quality of life for all items.The item wording for the item pool consisting of ReQoL-39 items is presented in Table Given the ordered categorical nature of the response categories, the Graded Response Model (GRM)  was appl2) [2 statistic is calculated for each item, one weakness in this approach is that it may lead to spurious results with large numbers of items. To reduce the impact of multiple testing we used a cross-validation approach [Model fit was evaluated by the sum-score-based item fit statistic (S-X2) . Since tapproach : the samSupport for sufficient unidimensionality  enables Item and test information functions were generated and examined in detail to provide an indication of how well item pools for instrument versions could estimate person latent scale locations. The maximum value of the item information function for each item ranged from 0.63 to 3.87. To summarise the function for each item, we computed the mean latent score weighted by the IRT item information function, thus establishing the centre of the item information function for each item. Further, for each item we calculated the score range where the item information function was higher than 0.5. Finally, we calculated test information functions and standard errors of measurement for the total item pool, the ReQoL-10, and the ReQoL-20 scales; and we calculated the range where measurement precision was higher than a 0.9 (by converting the information to reliability level). IRT analyses used IRTPRO 3.0 for the GRM  and itemn = 2862) and we hypothesised that their quality of life would be lower than those receiving care in primary care and the voluntary sector recruited from GP surgeries and charities, respectively (n = 1404). We used student’s t tests to assess the level of significance at 5%. We then compared the EAP scores for participants with different levels of self-reported general health and mental health, using one-way analysis of variance (ANOVA) to assess the level of significance at 5%. A non-parametric test of trend for the ranks was performed across self-reported general health and mental health in five categories ranging from very poor to excellent. We hypothesised that quality of life would be higher as we move up along this range. These analyses were carried out in Stata 14 [In order to explore the known-group validity of the 39 items, we compared IRT Expected A Posteriori (EAP) score estimates  (using aStata 14 .2) larger than 2% [Differential item functioning (DIF) is said to be present when participants with the same score level  endorse items differently by virtue of some characteristics other than the variation due to their current health status, in terms of their ReQoL scale score. DIF with regard to age (continuous variable), gender, ethnicity (white and non-white), and diagnosis  was evaluated through ordinal logistic regression models . The sim than 2% . Hence wThe mean age for the 4266 participants was 47 years and the age range was 16–98 years; 55% were female. The distribution of self-reported major diagnostic groups was depression/anxiety (43%), psychotic disorders (15%), bipolar disorders (10%), and personality disorders (6%); severity ranged from mild to severe; 5% of the sample had no psychiatric diagnosis . Seven items showed poor fit (i.e. significant misfit) in two out of four subsamples, 11 items showed poor fit in one out of four subsamples, while 19 items did not show poor fit in any subsample. Table I felt happy’, ‘I felt hopeful about my future’, and ‘I felt in control of my life’ had information-weighted means around 0 (see plots in appendix 4 of the electronic supplementary file).The estimated IRT discrimination parameters ranged from 1.4 to 3.6 (Table I felt people did not want to be around me” (d-R2 = 0.010) and “I felt hopeful about my future” (d-R2 = 0.012). However, in no instance, the magnitude of DIF came close to the threshold of a d-R2 effect size value of 0.02 (see Table A1 and A2 in appendix 5 of the electronic supplementary file).In tests of DIF with regard to age, gender, ethnicity, and diagnosis several statistically significant instances of DIF were found. Importantly, with regard to magnitude of DIF, the two largest values were found in analyses of age DIF for the items “p < 0.01), indicating a lower quality of life, for those accessing secondary care as hypothesised. The mean IRT scores were − 0.22 for those in secondary care compared with 0.42 for those accessing care in other settings, suggesting that the items could distinguish between these two distinct groups of participants. The trend test shows that a trend in EAP scores existed across the ordered levels of self-reported health (p < 0.01). We also found that the mean EAP scores differed significantly (p < 0.01) among the different levels of self-reported general and mental health with the lowest EAP scores for those who reported poorest physical and mental health. The marginal reliability for response pattern scores of the 39 items was extremely high, at a value of 0.98. Graphical representations can illustrate the effective measurement range achieved across the range of ReQoL latent values. Figure When assessing known-group validity, as shown in Table In this second paper describing the psychometric evidence that was used in the development of the ReQoL measures, we have presented the results from the unidimensional IRT analyses, including item parameters (slopes and thresholds), centre of information, and fit statistics. These show that the ReQoL measures are well targeted to the population characteristics typical of mental health services users in England. First, there are no marked problems with limited scoring range and thus, no noteworthy floor or ceiling effects. Second, there is low measurement error across the score range for most users and high marginal reliability estimate for the item pool. Third, we have shown that the IRT scores distinguish as hypothesised, between groups defined by type of care settings and by self-assessed general or mental health. Those who were accessing treatment in secondary care had lower quality of life than those accessing treatments in primary care and the community in general; those with poorer self-reported physical and mental health had a lower quality of life. Finally, none of the items showed troublesome differential item functioning.The unidimensional item parameters provided information to evaluate the ‘fit’ of the ReQoL items to the GRM, repeatedly in four independent and randomly selected samples. This was adopted because even minor item misfit is likely to be significant in a sample with over 4200 observations. This approach was rather conservative and we could have considered naming identifying items as poorly fitting (misfitting) if that was the case in two independent samples. The latter approach would have identified seven more items as poor, six of which were positively worded. It is noted that, at that stage, all the items identified as poorly fitting were retained because the misfit was not severe and also because the aim of this exercise was simply to assess the psychometric evidence (decisions were to be taken subsequently). The choice of items was subsequently made by the Scientific Group taking into consideration both the psychometric evidence and the qualitative evidence generated in early stages of the project .I could do the things I wanted to do’ was selected for the ReQoL-10 measure which was a “compromise” between psychometric accuracy and face validity, a collective decision made by service users, clinicians, and other experts.One of the two main purposes of these analyses was to provide strong psychometric parameters to choose the final forms for the ReQoL measures. Another paper has described the process where the evidence generated from this paper was combined with qualitative evidence on the items to produce ReQoL-10 and ReQoL-20 . A decisThe second purpose of the analyses was to use IRT analyses to provide the foundation for developing a utility measure based on the ReQoL-10 or ReQoL-20. The development of such a preference-based measure requires that different combinations of physical and mental health states are valued. In practice, only a subset of all possible health states can be assessed. Thus, to allow valuation of a reasonable number of health states, two steps are required: (1) further reduction in the number of items used to define health states, (2) for the final selection of items, identify the combination of item responses that are most likely to be encountered in practice. Commonly encountered health states should be valued directly, while utility values for rarely encountered combinations of health states may be derived by statistical modelling. Conventional approaches for selecting health states for valuation assume independence between items, and are inappropriate for ReQoL given the highly correlated items. Rasch analyses have become an increasingly popular method of construction health state classification systems –43 for ur = 0.98) and changes in IRT scores correlated strongly with sum score changes (r = 0.95). In known-group comparisons of primary and secondary care service users, IRT scores did not provide advantages in statistical power above the simple sum score [A limitation to this study is that the recruiting organisations were not chosen at random, nor were the individuals within organisations. However, given that participants were recruited from a number of organisations with a broad range of diagnoses, we are confident that the sample is representative of service users in the UK. In addition, the current scaling of the IRT score is defined by the current sample. Thus, 0 represents the mean of the mental health service users recruited for this study and the standard deviation of this sample is set to 1. Many recent applications of IRT methodology for patient-reported outcomes have used a representative general population sample to define the scale. Thus, in the patient-reported outcomes measurement information system (PROMIS) project, the mean of the general population has been set to 50 and the standard deviation to 10 . While gum score . FurtherThe IRT analyses suggest that the ReQoL item pool makes a coherent set for measuring the impact of mental problems on the lives of service users. Despite some limitations, the items provide precise measurement in the range where most service users are found and they were able to distinguish between different known groups. The results of IRT analyses have been used firstly, to provide the psychometric evidence to inform the item selection for the fixed form ReQoL-10 and ReQoL-20 questionnaires. Second, the analyses help establish the scoring of the intended continuum. Third, the results will be fed forward to the construction of the health state classification using a subset of the ReQoL-10 items to select health states with a view to eliciting preference weights from members of the general population. These steps when completed will make the ReQoL a preference-based outcome measure for calculating quality-adjusted life years as well as a stand-alone PROM.Supplementary file1 (DOCX 2321 kb)Below is the link to the electronic supplementary material.