Challenges and perspectives for naming lipids in the context of lipidomics.

INTRODUCTION: Lipids are key compounds in the study of metabolism and are increasingly studied in biology projects. It is a very broad family that encompasses many compounds, and the name of the same compound may vary depending on the community where they are studied. OBJECTIVES: In addition, their structures are varied and complex, which complicates their analysis. Indeed, the structural resolution does not always allow a complete level of annotation so the actual compound analysed will vary from study to study and should be clearly stated. For all these reasons the identification and naming of lipids is complicated and very variable from one study to another, it needs to be harmonized. METHODS & RESULTS: In this position paper we will present and discuss the different way to name lipids (with chemoinformatic and semantic identifiers) and their importance to share lipidomic results. CONCLUSION: Homogenising this identification and adopting the same rules is essential to be able to share data within the community and to map data on functional networks.

(Re-)use and (re-)analysis of publicly available metabolomics data.

Metabolomics, the systematic measurement of small molecules (<1000 Da) in a given biological sample, is a fast-growing field with many different applications. In contrast to transcriptomics and proteomics, sharing of data is not as widespread in metabolomics, though more scientists are sharing their data nowadays. However, to improve data analysis tools and develop new data analytical approaches and to improve metabolite annotation and identification, sharing of reference data is crucial. Here, different possibilities to share (metabolomics) data are reviewed and some recent approaches and applications regarding the (re-)use and (re-)analysis are highlighted.

Lipidomic and Metallomic Alteration of Caenorhabditis elegans after Acute and Chronic Manganese, Iron, and Zinc Exposure with a Link to Neurodegenerative Disorders.

Parkinson's disease (PD) progresses with the loss of dopaminergic neurons in the substantia nigra pars compacta region of the brain. The superior mechanisms and the cause of this specific localized neurodegeneration is currently unknown. However, experimental evidence indicates a link between PD progression and reactive oxygen species with imbalanced metal homeostasis. Wild-type Caenorhabditis elegans exposed to redox-active metals was used as the model organism to study cellular response to imbalanced metal homeostasis linked to neurodegenerative diseases. Using modern hyphenated techniques such as capillary electrophoresis coupled to inductively coupled plasma mass spectrometry and ultrahigh-performance liquid chromatography mass spectrometry, alterations in the lipidome and metallome were determined in vivo. In contrast to iron, most of the absorbed zinc and manganese were loosely bound. We observed changes in the phospholipid composition for acute iron and manganese exposures, as well as chronic zinc exposure. Furthermore, we focused on the mitochondrial membrane alteration due to its importance in neuronal function. However, significant changes in the inner mitochondrial membrane by determination of cardiolipin species could only be observed for acute iron exposure. These results indicate different intracellular sites of local ROS generation, depending on the redox active metal. Our study combines metallomic and lipidomic alterations as the cause and consequence to enlighten intracellular mechanisms in vivo, associated with PD progression. The mass spectrometry raw data have been deposited to the MassIVE database (https://massive.ucsd.edu) with the identifier MSV000090796 and 10.25345/C51J97C8F.

APEX: an Annotation Propagation Workflow through Multiple Experimental Networks to Improve the Annotation of New Metabolite Classes in Caenorhabditis elegans.

Spectral similarity networks, also known as molecular networks, are crucial in non-targeted metabolomics to aid identification of unknowns aiming to establish a potential structural relation between different metabolite features. However, too extensive differences in compound structures can lead to separate clusters, complicating annotation. To address this challenge, we developed an automated Annotation Propagation through multiple EXperimental Networks (APEX) workflow, which integrates spectral similarity networks with mass difference networks and homologous series. The incorporation of multiple network tools improved annotation quality, as evidenced by high matching rates of the molecular formula derived by SIRIUS. The selection of manual annotations as the Seed Nodes Set (SNS) significantly influenced APEX annotations, with a higher number of seed nodes enhancing the annotation process. We applied APEX to different Caenorhabditis elegans metabolomics data sets as a proof-of-principle for the effective and comprehensive annotation of glycerophospho N-acyl ethanolamides (GPNAEs) and their glyco-variants. Furthermore, we demonstrated the workflow's applicability to two other, well-described metabolite classes in C. elegans, specifically ascarosides and modular glycosides (MOGLs), using an additional publicly available data set. In summary, the APEX workflow presents a powerful approach for metabolite annotation and identification by leveraging multiple experimental networks. By refining the SNS selection and integrating diverse networks, APEX holds promise for comprehensive annotation in metabolomics research, enabling a deeper understanding of the metabolome.

MobilityTransformR: an R package for effective mobility transformation of CE-MS data.

SUMMARY: We present MobilityTransformR, an R/Bioconductor package for the effective mobility scaling of capillary zone electrophoresis-mass spectrometry (CE-MS) data. It uses functionality from different R packages that are frequently used for data processing and analysis in MS-based metabolomics workflows, allowing the subsequent use of reproducible transformed CE-MS data in existing workflows. AVAILABILITY AND IMPLEMENTATION: MobilityTransformR is implemented in R (Version >= 4.2) and can be downloaded directly from the Bioconductor database (https://bioconductor.org/packages/MobilityTransformR) or GitHub (https://github.com/LiesaSalzer/MobilityTransformR). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Capillary electrophoresis-mass spectrometry as a tool for Caenorhabditis elegans metabolomics research.

INTRODUCTION: Polar metabolites in Caenorhabditis elegans (C. elegans) have predominantly been analyzed using hydrophilic interaction liquid chromatography coupled to mass spectrometry (HILIC-MS). Capillary electrophoresis coupled to mass spectrometry (CE-MS) represents another complementary analytical platform suitable for polar and charged analytes. OBJECTIVE: We compared CE-MS and HILIC-MS for the analysis of a set of 60 reference standards relevant for C. elegans and specifically investigated the strengths of CE separation. Furthermore, we employed CE-MS as a complementary analytical approach to study polar metabolites in C. elegans samples, particularly in the context of longevity, in order to address a different part of its metabolome. METHOD: We analyzed 60 reference standards as well as metabolite extracts from C. elegans daf-2 loss-of-function mutants and wild-type (WT) samples using HILIC-MS and CE-MS employing a Q-ToF-MS instrument. RESULTS: CE separations showed narrower peak widths and a better linearity of the estimated response function across different concentrations which is linked to less saturation of the MS signals. Additionally, CE exhibited a distinct selectivity in the separation of compounds compared to HILIC-MS, providing complementary information for the analysis of the target compounds. Analysis of C. elegans metabolites of daf-2 mutants and WT samples revealed significant alterations in shared metabolites identified through HILIC-MS, as well as the presence of distinct metabolites. CONCLUSION: CE-MS was successfully applied in C. elegans metabolomics, being able to recover known as well as identify novel putative biomarkers of longevity.

Autophagy compensates for defects in mitochondrial dynamics.

Compromising mitochondrial fusion or fission disrupts cellular homeostasis; however, the underlying mechanism(s) are not fully understood. The loss of C. elegans fzo-1MFN results in mitochondrial fragmentation, decreased mitochondrial membrane potential and the induction of the mitochondrial unfolded protein response (UPRmt). We performed a genome-wide RNAi screen for genes that when knocked-down suppress fzo-1MFN(lf)-induced UPRmt. Of the 299 genes identified, 143 encode negative regulators of autophagy, many of which have previously not been implicated in this cellular quality control mechanism. We present evidence that increased autophagic flux suppresses fzo-1MFN(lf)-induced UPRmt by increasing mitochondrial membrane potential rather than restoring mitochondrial morphology. Furthermore, we demonstrate that increased autophagic flux also suppresses UPRmt induction in response to a block in mitochondrial fission, but not in response to the loss of spg-7AFG3L2, which encodes a mitochondrial metalloprotease. Finally, we found that blocking mitochondrial fusion or fission leads to increased levels of certain types of triacylglycerols and that this is at least partially reverted by the induction of autophagy. We propose that the breakdown of these triacylglycerols through autophagy leads to elevated metabolic activity, thereby increasing mitochondrial membrane potential and restoring mitochondrial and cellular homeostasis.

Venoarterial Extracorporeal Membrane Oxygenation With or Without Advanced Intervention for Massive Pulmonary Embolism.

INTRODUCTION: Massive pulmonary embolism (MPE) is a rare but highly fatal condition. Our study's objective was to evaluate the association between advanced interventions and survival among patients with MPE treated with venoarterial extracorporeal membrane oxygenation (VA-ECMO). METHODS: This is a retrospective review of the Extracorporeal Life Support Organization (ELSO) registry data. We included adult patients with MPE who were treated with VA-ECMO during 2010-2020. Our Primary outcome was survival to hospital discharge; secondary outcomes were ECMO duration among survivors and rates of ECMO-related complications. Clinical variables were compared using the Pearson chi-square and Kruskal-Wallis H tests. RESULTS: We included 802 patients; 80 (10%) received SPE and 18 (2%) received CDT. Overall, 426 (53%) survived to discharge; survival was not significantly different among those treated with SPE or CDT on VA-ECMO (70%) versus VA-ECMO alone (52%) or SPE or CDT before VA-ECMO (52%). Multivariable regression found a trend towards increased survival among those treated with SPE or CDT while on ECMO (AOR 1.8, 95% CI 0.9-3.6), but no significant correlation. There was no association between advanced interventions and ECMO duration among survivors, or rates of ECMO-related complications. CONCLUSION: Our study found no difference in survival in patients with MPE who received advanced interventions prior to ECMO, and a slight non-significant benefit in those who received advanced interventions while on ECMO.

Providing metabolomics education and training: pedagogy and considerations.

BACKGROUND: Metabolomics is a highly multidisciplinary and non-standardised research field. Metabolomics researchers must possess and apply extensive cross-disciplinary content knowledge, subjective experience-based judgement, and the associated diverse skill sets. Accordingly, appropriate educational and training initiatives are important in developing this knowledge and skills base in the metabolomics community. For these initiatives to be successful, they must consider both pedagogical best practice and metabolomics-specific contextual challenges. AIM OF REVIEW: The aim of this review is to provide consolidated pedagogical guidance for educators and trainers in metabolomics educational and training programmes. KEY SCIENTIFIC CONCEPTS OF REVIEW: In this review, we discuss the principles of pedagogical best practice as they relate to metabolomics. We then discuss the challenges and considerations in developing and delivering education and training in metabolomics. Finally, we present examples from our own teaching practice to illustrate how pedagogical best practice can be integrated into metabolomics education and training programmes.

Critical assessment of chromatographic metadata in publicly available metabolomics data repositories.

INTRODUCTION: The structural identification of metabolites represents one of the current bottlenecks in non-targeted liquid chromatography-mass spectrometry (LC-MS) based metabolomics. The Metabolomics Standard Initiative has developed a multilevel system to report confidence in metabolite identification, which involves the use of MS, MS/MS and orthogonal data. Limitations due to similar or same fragmentation pattern (e.g. isomeric compounds) can be overcome by the additional orthogonal information of the retention time (RT), since it is a system property that is different for each chromatographic setup. OBJECTIVES: In contrast to MS data, sharing of RT data is not as widespread. The quality of data and its (re-)useability depend very much on the quality of the metadata. We aimed to evaluate the coverage and quality of this metadata from public metabolomics repositories. METHODS: We acquired an overview on the current reporting of chromatographic separation conditions. For this purpose, we defined the following information as important details that have to be provided: column name and dimension, flow rate, temperature, composition of eluents and gradient. RESULTS: We found that 70% of descriptions of the chromatographic setups are incomplete (according to our definition) and an additional 10% of the descriptions contained ambiguous and/or incorrect information. Accordingly, only about 20% of the descriptions allow further (re-)use of the data, e.g. for RT prediction. Therefore, we have started to develop a unified and standardized notation for chromatographic metadata with detailed and specific description of eluents, columns and gradients. CONCLUSION: Reporting of chromatographic metadata is currently not unified. Our recommended suggestions for metadata reporting will enable more standardization and automatization in future reporting.

Quality assurance and quality control reporting in untargeted metabolic phenotyping: mQACC recommendations for analytical quality management.

BACKGROUND: Demonstrating that the data produced in metabolic phenotyping investigations (metabolomics/metabonomics) is of good quality is increasingly seen as a key factor in gaining acceptance for the results of such studies. The use of established quality control (QC) protocols, including appropriate QC samples, is an important and evolving aspect of this process. However, inadequate or incorrect reporting of the QA/QC procedures followed in the study may lead to misinterpretation or overemphasis of the findings and prevent future metanalysis of the body of work. OBJECTIVE: The aim of this guidance is to provide researchers with a framework that encourages them to describe quality assessment and quality control procedures and outcomes in mass spectrometry and nuclear magnetic resonance spectroscopy-based methods in untargeted metabolomics, with a focus on reporting on QC samples in sufficient detail for them to be understood, trusted and replicated. There is no intent to be proscriptive with regard to analytical best practices; rather, guidance for reporting QA/QC procedures is suggested. A template that can be completed as studies progress to ensure that relevant data is collected, and further documents, are provided as on-line resources. KEY REPORTING PRACTICES: Multiple topics should be considered when reporting QA/QC protocols and outcomes for metabolic phenotyping data. Coverage should include the role(s), sources, types, preparation and uses of the QC materials and samples generally employed in the generation of metabolomic data. Details such as sample matrices and sample preparation, the use of test mixtures and system suitability tests, blanks and technique-specific factors are considered and methods for reporting are discussed, including the importance of reporting the acceptance criteria for the QCs. To this end, the reporting of the QC samples and results are considered at two levels of detail: "minimal" and "best reporting practice" levels.

N-Alkylpyridinium sulfonates for retention time indexing in reversed-phase-liquid chromatography-mass spectrometry-based metabolomics.

Chromatographic retention time information is valuable, orthogonal information to MS and MS/MS data that can be used in metabolite identification. However, while comparison of MS data between different instruments is possible to a certain degree, retention times (RTs) can vary extensively, even when nominally the same phase system is used. Different factors such as column dead volumes, system extra column volume, and gradient dwell volume can influence absolute retention times. Retention time indexing (RTI), routinely employed in gas chromatography (e.g., Kovats index), allows compensation for deviations in experimental conditions. Different systems have been reported for RTI in liquid chromatography, but none of them have been applied to metabolomics to the same extent as they have with GC. Recently, a more universal RTI system has been reported based on a homologous series of N-alkylpyridinium sulfonates (NAPS). These reference standards ionize in both positive and negative ionization modes and are UV-active. We demonstrate the NAPS can be used for retention time indexing in reversed-phase-liquid chromatography-mass spectrometry (RP-LC-MS)-based metabolomics. Having measured >500 metabolite standards and varying flow rate and column dimension, we show that conversion of RT to retention indices (RI) substantially improves comparability of retention information and enables to use of RI for metabolite annotation and identification. Graphical Abstract.

WHEN AND HOW TO USE ORTHOSTATIC VITAL SIGNS.

BACKGROUND: Much controversy surrounds the use of orthostatic vital signs (OVS), including their indications, performance, and interpretation. This can lead to conflict between nurses, physicians, and consultants. This article summarizes the evidence for OVS in selected emergency department (ED) indications and the basis for a rapid measurement protocol. OBJECTIVE: This narrative review is intended to clarify indications for OVS measurement, their performance, and interpretation. DISCUSSION: Phlebotomy studies indicate that OVS are more discriminating than supine vital signs in hypovolemia, but many findings, even some considered "positive," do not provide compelling evidence in favor of or against disease. Evaluated as a diagnostic test, they have a low yield and controversial criteria for a positive test, but as vital signs, they are useful for selected patients with frequent ED presentations-blood loss, dehydration, dizziness, weakness, and falls. Available evidence supports a rapid measurement protocol, including a 1-min interval after standing. CONCLUSION: OVS are useful in selected patients, in a variety of frequent presentations, but their indications and implications for a patient's care are subject to physician interpretation. Given their ease of measurement and effect on decision-making, physicians may consider measuring them early in the evaluation of selected patients.

Early Prediction of Intensive Care Admission in Emergency Department Patients With Asthma.

BACKGROUND: Emergency physicians must choose whether patients with asthma are admitted to a hospital ward or a higher level of care, such as an intermediate care unit (IMC) or intensive care unit (ICU). OBJECTIVE: This study aimed to determine which variables, available early during emergency department (ED) visits, are associated with IMC/ICU admission. METHODS: In this retrospective chart review (records from 2015-2018), two trained abstractors, blinded to study hypothesis, abstracted data on predictor variables and disposition (ward vs. IMC/ICU). Predictor variables were defined explicitly and abstracted from the periods of ED arrival and after treatment with 7.5 mg nebulized albuterol. Distress was defined as tripod positioning or speaking in broken sentences. "Arrival" and "after treatment" scoring systems were derived based on adjusted odds ratios (aOR) for predictor variables. We performed analyses using SASⓇ, version 9.4 (SAS Institute). RESULTS: Among 273 patients, 105 required admission to an IMC/ICU. At presentation, distress (aOR 2.1, 95% confidence interval [CI] 1.1-3.9), room air SpO2 ≥95% (aOR 0.29, 95% CI 0.14-0.62), respiratory rate > 20 breaths/min (aOR 1.9, 95% CI 1.0-3.3), and retractions (aOR 1.9, 95% CI 1.1-3.3) were associated with IMC/ICU admission. After initial bronchodilator therapy, heart rate > 120 beats/min (aOR 7.1, 95% CI 2.0-25), room air SpO2 ≥ 95% (aOR 0.15, 95% CI 0.07-0.34), and noninvasive ventilation (aOR 6.5, 95% CI 2.5-17) were associated with IMC/ICU admission. Both scoring systems stratified risk of IMC/ICU admission into low-risk (9-10%) and high-risk (70-100%) groups. CONCLUSIONS: Combinations of predictor variables, available early in a patient's stay, stratify risk of admission to an IMC/ICU bed.

Quantitative and Qualitative Analysis of Emergency Physician Performed Biparietal Diameter Estimate for Gestational Age.

BACKGROUND: An accurate estimation of fetal gestational age is essential for the management of pregnant patients who present to the emergency department (ED). Point-of-care-ultrasound (POCUS) is an integral part of emergency medicine training and includes measurement of fetal gestational age by the biparietal diameter (BPD) method. OBJECTIVES: In this study we performed a quantitative assessment of emergency physician (EP)-performed BPD estimate of gestational age to identify the percentage of studies performed in our department that had an estimated gestational age within 14 days of an adjusted radiological or obstetrical estimation. METHODS: We performed a chart review of our ED ultrasound database and electronic medical records for cases where a BPD measurement was performed by an EP. We recorded the ED gestational age estimate in days and the radiological or obstetrical estimate of gestational age in days. We then calculated the difference in days between the two examinations. We used a normal binomial approximation to calculate 95% confidence intervals. A secondary analysis looked at the quality of the images based on measurement placement and the view obtained. RESULTS: Seventy-four cases met eligibility criteria; of those, 67 (91%) had a gestational age estimation within 14 days of the adjusted radiological or obstetrical estimate (95% confidence interval 81-96%). CONCLUSION: This study shows that EP-performed BPD measurements for gestational age are quantitatively accurate, with 91% of estimates within 14 days of a standard radiological or obstetrical estimation.

Comparison of lipidome profiles of Caenorhabditis elegans-results from an inter-laboratory ring trial.

INTRODUCTION: Lipidomic profiling allows 100s if not 1000s of lipids in a sample to be detected and quantified. Modern lipidomics techniques are ultra-sensitive assays that enable the discovery of novel biomarkers in a variety of fields and provide new insight in mechanistic investigations. Despite much progress in lipidomics, there remains, as for all high throughput "omics" strategies, the need to develop strategies to standardize and integrate quality control into studies in order to enhance robustness, reproducibility, and usability of studies within specific fields and beyond. OBJECTIVES: We aimed to understand how much results from lipid profiling in the model organism Caenorhabditis elegans are influenced by different culture conditions in different laboratories. METHODS: In this work we have undertaken an inter-laboratory study, comparing the lipid profiles of N2 wild type C. elegans and daf-2(e1370) mutants lacking a functional insulin receptor. Sample were collected from worms grown in four separate laboratories under standardized growth conditions. We used an UPLC-UHR-ToF-MS system allowing chromatographic separation before MS analysis. RESULTS: We found common qualitative changes in several marker lipids in samples from the individual laboratories. On the other hand, even in this controlled experimental system, the exact fold-changes for each marker varied between laboratories. CONCLUSION: Our results thus reveal a serious limitation to the reproducibility of current lipid profiling experiments and reveal challenges to the integration of such data from different laboratories.

UHPLC-IM-Q-ToFMS analysis of maradolipids, found exclusively in Caenorhabditis elegans dauer larvae.

Lipid identification is one of the current bottlenecks in lipidomics and lipid profiling, especially for novel lipid classes, and requires multidimensional data for correct annotation. We used the combination of chromatographic and ion mobility separation together with data-independent acquisition (DIA) of tandem mass spectrometric data for the analysis of lipids in the biomedical model organism Caenorhabditis elegans. C. elegans reacts to harsh environmental conditions by interrupting its normal life cycle and entering an alternative developmental stage called dauer stage. Dauer larvae show distinct changes in metabolism and morphology to survive unfavorable environmental conditions and are able to survive for a long time without feeding. Only at this developmental stage, dauer larvae produce a specific class of glycolipids called maradolipids. We performed an analysis of maradolipids using ultrahigh performance liquid chromatography-ion mobility spectrometry-quadrupole-time of flight-mass spectrometry (UHPLC-IM-Q-ToFMS) using drift tube ion mobility to showcase how the integration of retention times, collisional cross sections, and DIA fragmentation data can be used for lipid identification. The obtained results show that combination of UHPLC and IM separation together with DIA represents a valuable tool for initial lipid identification. Using this analytical tool, a total of 45 marado- and lysomaradolipids have been putatively identified and 10 confirmed by authentic standards directly from C. elegans dauer larvae lipid extracts without the further need for further purification of glycolipids. Furthermore, we putatively identified two isomers of a lysomaradolipid not known so far.

Clinically relevant adverse cardiovascular events in intermediate heart score patients admitted to the hospital following a negative emergency department evaluation.

STUDY HYPOTHESIS: Study objective: To estimate the frequency of clinically relevant adverse cardiac events (CRACE) in patients admitted to the hospital for chest pain with an intermediate HEART score (4, 5, 6), non-diagnostic EKG, and a negative initial troponin. METHODS: We conducted a retrospective analysis of all patients admitted to the University of Maryland Medical Center (UMMC) from May 2016 to May 2019 with an intermediate HEART score (4, 5, or 6), a non-diagnostic EKG, and a negative initial troponin. Our primary outcome was the rate of inpatient clinically relevant adverse cardiac events (CRACE), composite of life-threatening dysrhythmia, inpatient STEMI, cardiac or respiratory arrest, and all-cause mortality during hospitalization. RESULTS: A total of 1118 patients met our inclusion criteria, 6 of whom had CRACE. Overall the rate of CRACE was 0.5% (95% CI, 0.2-1.2%). Six patients (0.5%, 95% CI, 0.2%-1.2%) experienced inpatient NSTEMIs, 212 patients (19%, 95% CI, 17-21%) underwent provocative testing during their inpatient stay, 5 patients received a stent or CABG, and 5 patients had false positive non-invasive testing and underwent a negative cardiac catheterization. CONCLUSIONS: In this cohort of admitted patients with a documented intermediate-risk HEART score, nonischemic EKG, and negative initial troponin, the occurrence of CRACE during the index hospitalization was 0.5%.

Emergency Department Asthma "Spacing Trials": Institutional Variability and Time Cost.

BACKGROUND: Some admitting physicians request a medication-free interval ("spacing trial") in the emergency department (ED) to determine whether a patient with an acute exacerbation of asthma can be safely admitted to a hospital ward bed, where bronchodilators are only available every 4 h. OBJECTIVE: Our objectives were to estimate the frequency of ED spacing trials in different hospitals and their associated time cost. METHODS: This multicenter retrospective cohort study examined patients admitted for asthma from 2015 to 2018. We included all university records and a random sample of records from two community hospitals in the same urban area. Two team members abstracted data from each record using recommended methods, with group consensus to resolve differences. Proportion confidence intervals were calculated using normal binomial approximation. We calculated mean differences in ED stay associated with spacing trials, using multivariable linear regression to adjust for age, hospital type, history of intubation, initial pulse, initial respiratory rate, initial signs of distress. RESULTS: We collected data from 274 patients in the university hospital, and 71 and 70 cases from the community hospitals. An explicit spacing trial was noted in 52 of 274 (19%) university hospital records vs. 3 of 141 (2%) community hospital records, with a difference of 17% (95% confidence interval [CI] 11-23%). Delayed patient decompensation occurred in 3%, with no difference between hospitals. Spacing trials were associated with an adjusted mean of 159 min (95% CI 102-217 min) increase in ED stay. CONCLUSIONS: The practice of spacing varies widely between hospitals and is associated with substantial delay without an apparent benefit.