Cell Labeling with Magneto-Endosymbionts and the Dissection of the Subcellular Location,Fate, and Host Cell Interactions

Purpose

The purposes of this study are to characterize magneto-endosymbiont (ME) labeling of mammalian cells and to discern the subcellular fate of these living contrast agents. MEs are novel magnetic resonance imaging (MRI) contrast agents that are being used for cell tracking studies. Understanding the fate of MEs in host cells is valuable for designing in vivo cell tracking experiments.

Procedures

The ME’s surface epitopes, contrast-producing paramagnetic magnetosomal iron, and genome were studied using immunocytochemistry (ICC), Fe and MRI contrast measurements, and quantitative polymerase chain reaction (qPCR), respectively. These assays, coupled with other common assays, enabled validation of ME cell labeling and dissection of ME subcellular processing.

Results

The assays mentioned above provide qualitative and quantitative assessments of cell labeling, the subcellular localization and the fate of MEs. ICC results, with an ME-specific antibody, qualitatively shows homogenous labeling with MEs. The ferrozine assay shows that MEs have an average of 7 fg Fe/ME, ～30 % of which contributes to MRI contrast and ME-labeled MDA-MB-231 (MDA-231) cells generally have 2.4 pg Fe/cell, implying ～350 MEs/cell. Adjusting the concentration of Fe in the ME growth media reduces the concentration of non-MRI contrast-producing Fe. Results from the qPCR assay, which quantifies ME genomes in labeled cells, shows that processing of MEs begins within 24 h in MDA-231 cells. ICC results suggest this intracellular digestion of MEs occurs by the lysosomal degradation pathway. MEs coated with listeriolysin O (LLO) are able to escape the primary phagosome, but subsequently co-localize with LC3, an autophagy-associated molecule, and are processed for digestion. In embryos, where autophagy is transiently suppressed, MEs show an increased capacity for survival and even replication. Finally, transmission electron microscopy (TEM) of ME-labeled MDA-231 cells confirms that the magnetosomes (the MRI contrast-producing particles) remain intact and enable in vivo cell tracking.

Conclusions

MEs are used to label mammalian cells for the purpose of cell tracking in vivo, with MRI. Various assays described herein (ICC, ferrozine, and qPCR) allow qualitative and quantitative assessments of labeling efficiency and provide a detailed understanding of subcellular processing of MEs. In some cell types, MEs are digested, but the MRI-producing particles remain. Coating with LLO allows MEs to escape the primary phagosome, enhances retention slightly, and confirms that MEs are ultimately processed by autophagy. Numerous intracellular bacteria and all endosymbiotically derived organelles have evolved molecular mechanisms to avoid intracellular clearance, and identification of the specific processes involved in ME clearance provides a framework on which to develop MEs with enhanced retention in mammalian cells.

     

Computerized physician order entry and decision support improves ED analgesic ordering for renal colic

Objectives

Computerized physician order entry (CPOE) offers the potential for safer, faster patient care, as well as greater use of evidence-based therapy via built-in decision support. However, the effectiveness of CPOE in yielding these benefits has shown mixed results in the emergency department (ED) setting. Our objective was to evaluate the impact of CPOE implementation on analgesic prescribing and dosing practices for renal colic presentations.

Methods

This retrospective pre/post comparative study was conducted in 3 tertiary hospitals that implemented CPOE in 2010. Two patient groups were compared: prior to (pre-CPOE) and after (post-CPOE) CPOE implementation. Each group consisted of 230 randomly selected, high-acuity patients presenting to the ED with renal colic. The primary outcome was the proportion of patients receiving ketorolac in the ED. Secondary outcomes included choice of analgesic and average morphine dose.

Results

The proportion of patients receiving ketorolac significantly increased after CPOE implementation (65.6% pre-CPOE vs 76.5% post-CPOE, P = .015), as did the proportion of patients receiving fentanyl (pre, 9.7%; post, 16.7%; P = .047). Differences in morphine use (pre, 66.0%; post, 69.1%) and average morphine dose (pre, 10.09 mg; post, 12.28 mg) did not reach statistical significance.

Conclusions

The introduction of CPOE is associated with an increase in ketorolac use for ED renal colic visits. This may reflect the inclusion of ketorolac in the renal colic order set. Computerized physician order entry implementation with condition-specific electronic order sets and decision support may improve evidence-based practice.     

Characterization of Magneto-Endosymbionts as MRI Cell Labeling and Tracking Agents

Purpose

Magneto-endosymbionts (MEs) show promise as living magnetic resonance imaging (MRI) contrast agents for in vivo cell tracking. Here we characterize the biomedical imaging properties of ME contrast agents, in vitro and in vivo.

Procedures

By adapting and engineering magnetotactic bacteria to the intracellular niche, we are creating magneto-endosymbionts (MEs) that offer advantages relative to passive iron-based contrast agents (superparamagnetic iron oxides, SPIOs) for cell tracking. This work presents a biomedical imaging characterization of MEs including: MRI transverse relaxivity (r ₂) for MEs and ME-labeled cells (compared to a commercially available iron oxide nanoparticle); microscopic validation of labeling efficiency and subcellular locations; and in vivo imaging of a MDA-MB-231BR (231BR) human breast cancer cells in a mouse brain.

Results

At 7T, r ₂ relaxivity of bare MEs was higher (250 s^?1 mM^?1) than that of conventional SPIO (178 s^?1 mM^?1). Optimized in vitro loading of MEs into 231BR cells yielded 1–4 pg iron/cell (compared to 5–10 pg iron/cell for conventional SPIO). r ₂ relaxivity dropped by a factor of ~3 upon loading into cells, and was on the same order of magnitude for ME-loaded cells compared to SPIO-loaded cells. In vivo, ME-labeled cells exhibited strong MR contrast, allowing as few as 100 cells to be detected in mice using an optimized 3D SPGR gradient-echo sequence.

Conclusions

Our results demonstrate the potential of magneto-endosymbionts as living MR contrast agents. They have r ₂ relaxivity values comparable to traditional iron oxide nanoparticle contrast agents, and provide strong MR contrast when loaded into cells and implanted in tissue.

     

Errors in fracture diagnoses in the emergency department – characteristics of patients and diurnal variation

Background

Evaluation of the circumstances related to errors in diagnosis of fractures at an Emergency Department may suggest ways to reduce the incidence of such errors.

Methods

Retrospective analysis of all cases during a two year period (2002–2004) where a fracture had been overlooked or an injury had been erroneously diagnosed as a fracture (n = 61). 100 random selected patients with correctly diagnosed fractures served as control group.

Results

In the two year period 5879 patients visited the ED with injuries. 1% of all visits to the ED resulted in an error in fracture diagnosis and 3.1% of all fractures were not diagnosed at the initial visit to the ED. 86% of such errors had consequences for treatment. No patient characteristics could be identified as risk factors for a misdiagnosis of a fracture. There was a peak in errors in fracture diagnoses between 8 pm and 2 am (47% against 20% in controls, p < 0.005).

Conclusion

A considerable number of fractures were not correctly diagnosed at the initial ED visit. There was a diurnal variation in the rate of misdiagnosis of fractures with a significant peak from 8 pm to 2 am. Where there was an error in fracture diagnosis, the patients did not appear to have a characteristic profile as regarding e.g. age, sex or capability to communicate with the ED staff. Increased consultancy service in radiology may reduce the frequency of errors in diagnosis, particularly in the evenings between 8 pm and 2 am.     

Using Computerized Provider Order Entry to Enforce Documentation of Tests with Pending Results at Hospital Discharge

Background

Small numbers of tests with pending results are documented in hospital discharge summaries leading to breakdown in communication and medical errors due to inadequate followup.

Objective

Evaluate effect of using a computerized provider order entry (CPOE) system to enforce documentation of tests with pending results into hospital discharge summaries.

Methods

We assessed the percent of all tests with pending results and those with actionable results that were documented before (n = 182 discharges) and after (n = 203 discharges) implementing the CPOE-enforcement tool. We also surveyed providers (n = 52) about the enforcement functionality.

Results

Documentation of all tests with pending results improved from 12% (87/701 tests) before to 22% (178/812 tests) (p = 0.02) after implementation. Documentation of tests with eventual actionable results increased from 0% (0/24) to 50% (14/28)(p<0.001). Survey respondents felt the intervention improved quality of summaries, provider communication, and was not time-consuming.

Conclusions

A CPOE tool enforcing documentation of tests with pending results into discharge summaries significantly increased documentation rates, especially of actionable tests. However, gaps in documentation still exist.     

Prospective evaluation of the treatment of pain in the ED using computerized physician order entry

Background

Treatment of pain in the emergency department (ED) is a significant area of focus, as previous studies have noted generally inadequate treatment of pain in ED patients. Previous studies have not evaluated the impact of computerized physician order entry (CPOE) on the treatment of pain in the ED. We sought to evaluate treatment of pain before and after implementation of CPOE in an academic ED.

Methods

We prospectively enrolled a convenience sample of patients presenting to the ED with a pain-related complaint in 4-month periods before and after CPOE implementation. We compared numbers who received pain medications, time from registration to administration of pain medication, and repeat dosing of pain medication.

Results

Six hundred forty-six ED patients participated in the pre-CPOE period, whereas 592 patients participated post-CPOE. Similar numbers of patients received pain medications in the pre-CPOE and post-CPOE periods (55% vs 59%; P = .139), whereas those in the post-CPOE period were more likely to receive a repeat dose of pain medications (10.5% vs 17.6%; P < .001).

Conclusion

The use of CPOE in the ED may offer modest benefits in the treatment of patients with pain-related complaints.     

Public opinion of a stroke clinical trial using exception from informed consent

Background

Acute stroke is a leading cause of morbidity and mortality. Clinical trials in stroke are challenging because victims often do not have the capacity to provide informed consent, excluding those patients most likely to benefit from the research.

Aim

We evaluated patient willingness to participate in a hypothetical acute stroke trial using an exception from informed consent.

Methods

Consecutive patients presenting to four emergency departments (EDs) underwent structured interviews regarding a hypothetical stroke trial using an exception from informed consent.

Results

Of 461 (72% of eligible) participants, 55% (95% CI, 50%–59%) were willing to be enrolled in the hypothetical study without giving informed consent. After multivariable analysis, independent predictors of willingness to enroll included Catholic religion (OR 1.57, 95% CI 1.17–2.10) and belief that current therapy offers a >50% chance of full recovery (OR 1.29, 95% CI 1.05–1.57). There was no difference between the proportion willing to enroll in a cardiac arrest study vs. a stroke study (55% vs. 55%, p?=?0.83)

Conclusions

Fifty-five percent of ED patients would be willing to be enrolled in a stroke trial using exception from informed consent.     

Intraoperative detection and localization of cylindrical implants in cone-beam CT image data

Purpose

Orthopedic fractures are often fixed using metal implants. The correct positioning of cylindrical implants such as surgical screws, rods and guide wires is highly important. Intraoperative 3D imaging is often used to ensure proper implant placement. However, 3D image interaction is time-consuming and requires experience. We developed an automatic method that simplifies and accelerates location assessment of cylindrical implants in 3D images.

Methods

Our approach is composed of three major steps. At first, cylindrical characteristics are detected by analyzing image gradients in small image regions. Next, these characteristics are grouped in a cluster analysis. The clusters represent cylindrical implants and are used to initialize a cylinder-to-image registration. Finally, the two end points are optimized regarding image contrast along the cylinder axis.

Results

A total of 67 images containing 420 cylindrical implants were used for testing. Different anatomical regions (calcaneus, spine) and various image sources (two mobile devices, three reconstruction methods) were investigated. Depending on the evaluation set, the detection performance was between 91.7 and 96.1 % true- positive rate with a false-positive rate between 2.0 and 3.2 %. The end point distance errors ranged from \(1.0 \pm 1.2\) to \(4.3 \pm 2.9\)  mm and the orientation errors from \(1.6 \pm 2.2\) to \(2.3 \pm 2.2\) degrees. The average computation time was less than 5 seconds.

Conclusions

An automatic method was developed and tested that obviates the need for 3D image interaction during intraoperative assessment of cylindrical orthopedic implants. The required time for working with the viewing software of cone-beam CT device is drastically reduced and leads to a shorter time under anesthesia for the patient.     

Small dead space heat and moisture exchangers do not impede gas exchange during noninvasive ventilation: a comparison with a heated humidifier

Objective

Adverse respiratory and gasometrical effects have been described in patients with acute respiratory failure (ARF) undergoing noninvasive ventilation (NIV) with standard heat and moisture exchangers (HME). We decided to evaluate respiratory parameters and arterial blood gases (ABG) of patients during NIV with small dead space HME compared with heated humidifier (HH).

Design

Prospective randomized crossover study.

Setting

A 16-bed medical intensive care unit (ICU).

Patients

Fifty patients receiving NIV for ARF.

Measurements

The effects of HME and HH on respiratory rate, minute ventilation, EtCO₂, oxygen saturation, airway occlusion pressure at 0.1 s, ABG, and comfort perception were compared during two randomly determined NIV periods of 30 min. The relative impact of HME and HH on these parameters was successively compared with or without addition of a flex tube (40 and 10 patients, respectively).

Main results

No difference was observed between HME and HH regarding any of the studied parameters, whether or not a flex tube was added.

Conclusion

If one decides to humidify patients’ airways during NIV, one may do so with small dead space HME or HH without altering respiratory parameters.     

Response to Medication Dosing Alerts for Pediatric Inpatients Using a Computerized Provider Order Entry System

Objective

Medication dosing errors are of particular concern in hospitalized children. Avoidance of such errors is essential to quality improvement and patient safety. Computerized provider order entry (CPOE) systems with clinical decision support (CDS) have the potential to reduce medication errors. The objective of this study was to evaluate provider response to the dosing alerts in a CPOE system with CDS for pediatric inpatients and to identify differences in provider response based on clinician specialty.

Patients and methods

We conducted a retrospective analysis of all medication dosing alerts over a 1-year period (January 1 through December 31, 2008) for all pediatric inpatients at Hospital for Special Surgery. Alerts were analyzed with respect to medication dosing, prescriber, and action taken by the prescriber after the alert was triggered (i.e., accepted suggested change, ignored recommendation/overrode, or cancelled the order).

Results

During the study period, 18,163 medication orders were placed and 1,024 dosing alerts were fired. Overdosing of medications accounted for 91% of the alerts and underdosing 9%. The pediatric-trained providers ignored more alerts and cancelled fewer orders than the non-pediatric-trained providers (p<0.001). Both groups changed the order similarly based on CDS recommendations.

Conclusions

Differences in response to CDS were found between pediatric-trained and non-pediatric-trained providers caring for pediatric patients; however, both groups changed orders based on CDS similarly. CPOE with built-in CDS may be of particular value when providers with different specialties and types of training are caring for pediatric patients.     

Three-dimensional prediction of free-flap volume in autologous breast reconstruction by CT angiography imaging

Purpose

   The diagnostic use of computer tomography angiography (CTA) to identify perforating blood vessels for abdominal free-flap breast reconstruction was extended to estimate the three-dimensional (3D) preoperative flap volume and to compare it with the real intraoperative flap weights in order to (1) evaluate the accuracy of CTA-based 3D flap volume prediction, and (2) to analyze abdominal tissue estimation for required breast volume reconstruction.

Methods

   Preoperative CTA was performed in 54 patients undergoing unilateral breast reconstruction with a free, deep, inferior epigastric artery perforator flap. 3D flap volumes ( \(\hbox {cm}^{3}\) ) based on CTA data were calculated and compared with the actual intraoperative flap weight (g). In addition, a breast volume to flap volume ratio was calculated to analyze whether the estimated 3D abdominal flap volume would match that of the breast to be removed.

Results

   40 CTA data sets (74.1 %) fulfilled the technical requirements for a reliable determination of flap volume. 3D CTA flap volume prediction showed no relevant differences to the actual flap weight (p = 0.44) and high correlations (r = 0.998, \(p < 0.001\) ), allowing a prediction accuracy within 0.29 \(\pm \) 3.0 % (range: from \(-\) 8.77 to 5.67 %) of the real flap weight. Significantly larger flap volumes were harvested compared with the actually required breast volumes ( \(p < 0.001\) ), leading to an average of 21 % of the remnant flap tissue potentially being discarded.

Conclusions

   CTA-based 3D flap volume prediction provides accurate preoperative guidelines concerning the needed amount of abdominal tissue that can be harvested to achieve acceptable symmetry.     

Optimized order estimation for autoregressive models to predict respiratory motion

Purpose

To successfully ablate moving tumors in robotic radio-surgery, it is necessary to compensate for motion of inner organs caused by respiration. This can be achieved by tracking the body surface and correlating the external movement with the tumor position as it is implemented in the CyberKnife $^\circledR $ Synchrony system. Tracking errors, originating from system immanent time delays, are typically reduced by time series prediction. Many prediction algorithms exploit autoregressive (AR) properties of the signal. Estimating the optimal model order $p$ for these algorithms constitutes a challenge often solved via grid search or prior knowledge about the signal.

Methods

Aiming at a more efficient approach instead, this study evaluates the Akaike information criterion (AIC), the corrected AIC, and the Bayesian information criterion (BIC) on the first minute of the respiratory signal. Exemplarily, we evaluated the approach for a least mean square (LMS) and a wavelet-based LMS (wLMS) predictor.

Results

Analyzing 12 motion traces, orders estimated by AIC had the highest prediction accuracy for both prediction algorithms. Extending the investigations to 304 real motion traces, the prediction error of wLMS using AIC was found to decrease significantly by 85.1 % of the data compared to the original implementation

Conclusions

The overall results suggest that using AIC to estimate the model order $p$ for prediction algorithms based on AR properties is a valid method which avoids intensive grid search and leads to high prediction accuracy.     

Simultaneous extraction of carotid artery intima-media interfaces in ultrasound images: assessment of wall thickness temporal variation during the cardiac cycle

Objectives

   The aim of this work is to present and evaluate a novel segmentation method for localizing the contours of the intima-media complex in the carotid artery wall through longitudinal ultrasound B-mode imaging. The method is used to investigate the association between atherosclerosis risk factors and the cyclic variation of the intima-media thickness during the heart beat.

Methods

   The framework introduced is based on two main features. The first is a simultaneous extraction of both the lumen-intima and the media-adventitia interfaces, using the combination of an original shape-adapted filter bank and a specific dynamic programming scheme. The second is an innovative spatial transformation that eases the extraction of skewed and curved contours, and exploits the result from the previous image as a priori information, when processing the current image. The intima-media thickness is automatically derived from the estimated contours for each time step during the cardiac cycle. Our method was evaluated in vivo on 57 healthy volunteers and 25 patients at high cardiovascular risk. Reference contours were generated for each subject by averaging the tracings performed by three experienced observers.

Results

   Segmentation errors were \(29 \pm 27\,\upmu \hbox {m}\) for the lumen-intima interface, \(42 \pm 38\,\upmu \hbox {m}\) for the media-adventitia interface, and \(22 \pm 16\,\upmu \hbox {m}\) for the intima-media thickness. This uncertainty was similar to inter- and intra-observer variability. Furthermore, the amplitude of the temporal variation in thickness of the intima-media layers during the cardiac cycle was significantly higher in at-risk patients compared to healthy volunteers \((79 \pm 36\) vs. \(64 \pm 26\,\upmu \hbox {m},\, p=0.032)\) .

Conclusion

   The method proposed may provide a relevant diagnostic aid for atherosclerosis screening in clinical studies.