Characterizing and Managing Missing Structured Data in Electronic Health Records: Data Analysis.

BACKGROUND: Missing data is a challenge for all studies; however, this is especially true for electronic health record (EHR)-based analyses. Failure to appropriately consider missing data can lead to biased results. While there has been extensive theoretical work on imputation, and many sophisticated methods are now available, it remains quite challenging for researchers to implement these methods appropriately. Here, we provide detailed procedures for when and how to conduct imputation of EHR laboratory results. OBJECTIVE: The objective of this study was to demonstrate how the mechanism of missingness can be assessed, evaluate the performance of a variety of imputation methods, and describe some of the most frequent problems that can be encountered. METHODS: We analyzed clinical laboratory measures from 602,366 patients in the EHR of Geisinger Health System in Pennsylvania, USA. Using these data, we constructed a representative set of complete cases and assessed the performance of 12 different imputation methods for missing data that was simulated based on 4 mechanisms of missingness (missing completely at random, missing not at random, missing at random, and real data modelling). RESULTS: Our results showed that several methods, including variations of Multivariate Imputation by Chained Equations (MICE) and softImpute, consistently imputed missing values with low error; however, only a subset of the MICE methods was suitable for multiple imputation. CONCLUSIONS: The analyses we describe provide an outline of considerations for dealing with missing EHR data, steps that researchers can perform to characterize missingness within their own data, and an evaluation of methods that can be applied to impute clinical data. While the performance of methods may vary between datasets, the process we describe can be generalized to the majority of structured data types that exist in EHRs, and all of our methods and code are publicly available.

Two-photon singlet oxygen microscopy: the challenges of working with single cells.

A microscope is described in which singlet molecular oxygen, O2(a1deltag), is produced in a femtoliter focal volume via a nonlinear two-photon photosensitized process, and the 1270 nm phosphorescence from this population of O2(a1deltag) is detected in a photon counting experiment. Although two-photon excitation of a sensitizer is less efficient than excitation by a one-photon process, nonlinear excitation has several distinct advantages with respect to the spatial resolution accessible. Pertinent aspects of this two-photon O2(a1deltag) microscope were characterized using bulk solutions of photosensitizers. These data were compared to those obtained from a single biological cell upon linear one-photon excitation of a sensitizer incorporated in the cell. On the basis of the results obtained, we outline the challenges of using nonlinear optical techniques to create O2(aldeltag) at the single cell level and to then optically detect the O2(aldeltag) thus produced in a time-resolved experiment.

5,10,15,20-tetrakis(N-methyl-4-pyridyl)-21H,23H-porphine (TMPyP) as a sensitizer for singlet oxygen imaging in cells: characterizing the irradiation-dependent behavior of TMPyP in a single cell.

Singlet molecular oxygen, a1Delta(g), can be detected from a single cell by its weak 1270 nm phosphorescence (a1Delta(g)-->X3Sigma(g)-) upon irradiation of the photosensitizer 5,10,15,20-tetrakis(N-methyl-4-pyridyl)-21H,23H-porphine (TMPyP) incorporated into the cell. The behavior of this sensitizer in a cell, and hence the behavior of the associated singlet oxygen phosphorescence signal, depends on the conditions under which the sample is exposed to light. Upon irradiation of a neuron freshly incubated with TMPyP, the intensity of TMPyP fluorescence initially increases and there is a concomitant increase in the singlet oxygen phosphorescence intensity from the cell. These results appear to reflect a photoinduced release of TMPyP bound to DNA in the nucleus of the cell, where TMPyP tends to localize, and the subsequent relocalization of TMPyP to a different microenvironment in the cell. Upon prolonged irradiation of the cell, TMPyP photobleaches and there is a corresponding decrease in the singlet oxygen phosphorescence intensity from the cell. The data reported herein provide insight into key factors that can influence photosensitized singlet oxygen experiments performed on biological samples.

Photodynamic therapy: a means to enhanced drug delivery to tumors.

Using the photosensitizer 2-[1-hexyloxyethyl]-2-devinyl pyropheophorbide-a, we have determined that photodynamic therapy (PDT) can be used to facilitate the delivery of macromolecular agents. PDT regimens that use low fluences and fluence rates were the most successful. This effect was demonstrated for fluorescent microspheres with diameters ranging from 0.1 to 2 microm. Such treatment given immediately before administration of Doxil, a liposomally encapsulated formulation of doxorubicin with an average diameter of 0.1 microm, significantly enhanced its accumulation in transplanted murine Colo 26 tumors. The combination of PDT and Doxil led to a highly significant potentiation in tumor control without concomitant enhancement of systemic or local toxicity. Interestingly, concentration-effect modeling suggested that the enhanced cure rate was greater than what was predicted based on the increase in intratumor Doxil concentration. In summary, we have developed a novel PDT treatment that enhances the delivery and efficacy of macromolecule-based cancer therapies such as Doxil.

Choice of oxygen-conserving treatment regimen determines the inflammatory response and outcome of photodynamic therapy of tumors.

The rate of light delivery (fluence rate) plays a critical role in photodynamic therapy (PDT) through its control of tumor oxygenation. This study tests the hypothesis that fluence rate also influences the inflammatory responses associated with PDT. PDT regimens of two different fluences (48 and 128 J/cm(2)) were designed for the Colo 26 murine tumor that either conserved or depleted tissue oxygen during PDT using two fluence rates (14 and 112 mW/cm(2)). Tumor oxygenation, extent and regional distribution of tumor damage, and vascular damage were correlated with induction of inflammation as measured by interleukin 6, macrophage inflammatory protein 1 and 2 expression, presence of inflammatory cells, and treatment outcome. Oxygen-conserving low fluence rate PDT of 14 mW/cm(2) at a fluence of 128 J/cm(2) yielded approximately 70-80% tumor cures, whereas the same fluence at the oxygen-depleting fluence rate of 112 mW/cm(2) yielded approximately 10-15% tumor cures. Low fluence rate induced higher levels of apoptosis than high fluence rate PDT as indicated by caspase-3 activity and terminal deoxynucleotidyl transferase-mediated nick end labeling analysis. The latter revealed PDT-protected tumor regions distant from vessels in the high fluence rate conditions, confirming regional tumor hypoxia shown by 2-(2-nitroimidazol-1[H]-yl)-N-(3,3,3-trifluoropropyl) acetamide staining. High fluence at a low fluence rate led to ablation of CD31-stained endothelium, whereas the same fluence at a high fluence rate maintained vessel endothelium. The highest levels of inflammatory cytokines and chemokines and neutrophilic infiltrates were measured with 48 J/cm(2) delivered at 14 mW/cm(2) ( approximately 10-20% cures). The optimally curative PDT regimen (128 J/cm(2) at 14 mW/cm(2)) produced minimal inflammation. Depletion of neutrophils did not significantly change the high cure rates of that regimen but abolished curability in the maximally inflammatory regimen. The data show that a strong inflammatory response can contribute substantially to local tumor control when the PDT regimen is suboptimal. Local inflammation is not a critical factor for tumor control under optimal PDT treatment conditions.

Lifetime and diffusion of singlet oxygen in a cell.

In time- and spatially resolved experiments, singlet molecular oxygen, O(2)(a(1)Delta(g)), was created in a single nerve cell upon irradiation of a sensitizer incorporated in the cell nucleus using a focused laser beam. The singlet oxygen thus produced was detected by its infrared phosphorescence. Data obtained indicate that, contrary to common perception, this reactive species can be quite long-lived in a cell and, as such, can diffuse over appreciable distances including across the cell membrane into the extra-cellular environment. These results provide a new perspective for mechanistic studies of photoinduced cell death and intracellular signaling.

Direct optical detection of singlet oxygen from a single cell.

Singlet oxygen has been detected in single nerve cells by its weak 1270 nm phosphorescence (a1deltag --> X3sigmag-) upon irradiation of a photosensitizer incorporated in the cell. Thus, one can now consider the application of direct optical imaging techniques to mechanistic studies of singlet oxygen at the single-cell level.

Effects of medializing calcaneal osteotomy on Achilles tendon lengthening and plantar foot pressures.

Posterior tibial tendon insufficiency, or adult acquired flatfoot deformity, involves collapse of the longitudinal arch of the foot with ensuing changes in the bony architecture of the foot as well. While it is generally accepted that a medializing calcaneal osteotomy (MCO) is a very useful treatment for restoring the fallen arch, questions regarding the effects of this procedure upon plantar foot pressures and Achilles tendon length changes need to be answered. This study focuses on changes in plantar foot pressures and Achilles tendon length as the result of performing a MCO. Fourteen fresh-frozen cadaver legs were used to test the effects of MCO on Achilles tendon length changes 2 cm proximal to the Achilles tendon insertion on the calcaneus. Differential variable reluctance transducers were anchored in ventromedial, dorsomedial, dorsolateral, and ventrolateral positions of the Achilles tendon at the aforementioned level. The effects of the MCO on plantar foot pressures were assessed simultaneously using the Tekscan HR Mat. Axial loading (100 lbs) of each specimen was performed in neutral and dorsiflexion (15 degrees). Data were gathered for Achilles tendon length changes and plantar foot pressures for three trials in both the neutral and dorsiflexed positions. A medializing calcaneal osteotomy (1 cm medial translation) was then performed and testing was repeated in the fashion outlined heretofore. Analysis of the data revealed that there was no significant increase in Achilles tendon length as a result of the MCO. The data also showed that average pressure over the first and second metatarsal regions of the forefoot decreased significantly after MCO. At the same time there was a significant increase in average pressure over the medial and lateral aspect of the heel. These findings suggest that the Achilles tendon aids in inversion of the forefoot without undergoing a significant increase in length change of Achilles tendon fibers in any of the regions tested.

Effectiveness of levetiracetam in the treatment of lumbar radiculopathy: an open-label prospective cohort study.

OBJECTIVE: The purpose of this open-label study was to assess the effectiveness and tolerability of levetiracetam (LEV) in the treatment of patients with lower lumbar radiculopathy diagnosed by clinical presentation, physical examination, and electrodiagnostics. DESIGN: Open-label, forced titration, prospective cohort intent-to-treat analysis. SETTING: Major teaching hospital. PATIENTS: Participants were recruited from the university pain clinic and through referrals from the community. Subjects were eligible to participate if they (1) were older than 18 years, (2) had leg pain greater than back pain, (3) had been symptomatic for 6 months and <5 years, (3) had a baseline visual analog scale of at least 6 on the 0-10 scale, (4) had undergone lumbar spine magnetic resonance imaging within the last 12 months that showed no surgically correctable etiology for their radicular symptoms, and (5) had undergone electrodiagnostics that was positive for lumbar radiculopathy (L4, L5, or S1 nerve roots). Twenty-six patients were enrolled, and 24 (92.3%) completed the study. INTERVENTIONS: Patients initially received LEV 500 mg twice a day for 2 weeks. Dosages were then increased to 750 mg twice day for 2 weeks, and then to 1500 mg twice a day for the remainder of the study. Patients were observed for 8 weeks after reaching the maximum dose. MAIN OUTCOME MEASUREMENTS: Treatment effectiveness was assessed with the use of biweekly brief pain inventory and patient global assessment scales. Frequency and severity of adverse events were recorded. RESULTS: The mean biweekly worst and average pain scores decreased from 7.17 and 6.27 at baseline to 4.2 and 3.9, respectively, at week 12 (P < or = .001). Improvements were reported in general activity, ability to walk, mood, and relationship with people. Side effects consisted of sedation/drowsiness (46%), gastrointestinal upset (23%), headache (19%), blurred vision (15%), weakness/fatigue (11%), and dyscoordination (11%). CONCLUSIONS: This study showed that LEV might be a well-tolerated and effective treatment for lumbar radiculopathy. A large randomized placebo-controlled trial is recommended and comparative studies with alternative agents should be sought.

Control and selectivity of photosensitized singlet oxygen production: challenges in complex biological systems.

Singlet molecular oxygen is a reactive oxygen species that plays an important role in a number of biological processes, both as a signalling agent and as an intermediate involved in oxidative degradation reactions. Singlet oxygen is commonly generated by the so-called photosensitization process wherein a light-absorbing molecule, the sensitizer, transfers its energy of excitation to ground-state oxygen to make singlet oxygen. This process forms the basis of photodynamic therapy, for example, where light, a sensitizer, and oxygen are used to initiate cell death and ultimately destroy undesired tissue. Although the photosensitized production of singlet oxygen has been studied and used in biologically pertinent systems for years, the photoinitiated behaviour is often indiscriminate and difficult to control. In this Concept, we discuss new ideas and results in which spatial and temporal control of photosensitized singlet oxygen production can be implemented through the incorporation of the sensitizer into a conjugate system that selectively responds to certain triggers or stimuli.

Fluence rate as a modulator of PDT mechanisms.

BACKGROUND AND OBJECTIVES: Molecular oxygen in the tissue to be treated by photodynamic therapy (PDT) is critical for photodynamic cell killing. The fluence rate of PDT light delivery has been identified as an important modulator of tissue oxygenation and treatment outcome. This article provides supporting evidence for the role of fluence rate in PDT and discusses the underlying mechanisms. STUDY DESIGN/MATERIALS AND METHODS: Intratumoral pO2 was measured polarographically in murine tumors before and during PDT light treatment using the Eppendorf pO2 Histograph. Tumor response as a function of fluence rate and fluence was also assessed in murine tumor models. Changes in vascular permeability as a function of fluence rate were determined in murine tumors by measuring tumor uptake of fluorescent beads (200 nm diameter). RESULTS: Severe oxygen depletion is shown to occur within seconds of illumination at a fluence rate of 75 mW/cm2 in radiation-induced fibrosarcoma (RIF) tumors photosensitized with AlPcS2. This effect was reversible and consistent with photochemical oxygen depletion, which has been shown by us and others to be fluence rate dependent. It is demonstrated that fluence rate affects the PDT tumor response in the Colon 26 tumor model, high fluence rate diminishing or even totally inhibiting tumor control, low fluence rate promoting tumor control. The influence of fluence rate is not restricted to cytocidal effects, but can also be seen in sublethal conditions such as vascular permeability. CONCLUSIONS: Fluence rate of PDT light delivery exerts far-reaching control upon treatment outcome through its oxygenation modulating properties and possibly other mechanisms yet to be identified. This has been shown to be true in the preclinical and clinical setting. Further development of in situ dosimetry will be necessary to take full advantage of these discoveries.

DNA-programmed control of photosensitized singlet oxygen production.

DNA sequence-controlled on-and-off switching of a singlet oxygen sensitizer has been developed and demonstrated. The singlet oxygen photosensitizer pyropheophorbide-a (P) was attached to a 15-mer nucleotide sequence. A molecule that could quench the sensitizer, the so-called "black hole quencher 3" (Q), was attached to a complementary nucleotide strand. Upon hybridization of the two conjugates, singlet oxygen production from P was completely shut down. Upon the addition of a third DNA sequence that can displace and release the P-DNA conjugate from the P-Q pair, up to 85% of the singlet oxygen production was recovered. This system is a model for a benign drug that becomes active only in the presence of a specific targeted nucleotide sequence.

Optical detection of singlet oxygen from single cells.

The lowest excited electronic state of molecular oxygen, singlet molecular oxygen, O(2)(a (1)Delta(g)), is a reactive species involved in many chemical and biological processes. To better understand the roles played by singlet oxygen in biological systems, particularly at the sub-cellular level, optical tools have been developed to create and directly detect this transient state in time- and spatially-resolved experiments from single cells. Data obtained indicate that, contrary to common perception, this reactive species can be quite long-lived in a cell and, as such, can diffuse over appreciable distances including across the cell membrane into the extracellular environment. On one hand, these results demonstrate that the behavior of singlet oxygen in an intact cell can be significantly different from that inferred from model bulk studies. More generally, these results provide a new perspective for mechanistic studies of intra- and inter-cellular signaling and events that ultimately lead to photo-induced cell death.

Subcellular, time-resolved studies of singlet oxygen in single cells.

In time-resolved and spatially resolved experiments, singlet molecular oxygen, O2(a1Deltag), was created in a single nerve cell upon irradiation of a sensitizer incorporated in the cell using a focused laser beam. The singlet oxygen thus produced was detected by its infrared phosphorescence. Data obtained indicate that in both the cytoplasm and the nucleus of the cell, this reactive species is approximately 1-2 orders of magnitude longer-lived than previously believed. The data demonstrate that deactivation of singlet oxygen in the cell is dominated by interactions with the solvent not cellular constituents such as proteins. These results provide a new perspective for mechanistic studies of the role of O2(a1Deltag) in photoinduced cell death and intracellular signaling.

Singlet oxygen microscope: from phase-separated polymers to single biological cells.

The lowest excited electronic state of molecular oxygen, singlet molecular oxygen (a1Deltag), is an intermediate in many chemical and biological processes. Tools and methods have been developed to create singlet-oxygen-based optical images of heterogeneous samples that range from phase-separated polymers to biological cells. Such images provide unique insight into a variety of oxygen-dependent phenomena, including the photoinitiated death of cells.