Development and characterization of phasor-based analysis for FLIM to evaluate the metabolic and epigenetic impact of HER2 inhibition on squamous cell carcinoma cultures.

SIGNIFICANCE: Deranged metabolism and dysregulated growth factor signaling are closely associated with abnormal levels of proliferation, a recognized hallmark in tumorigenesis. Fluorescence lifetime imaging microscopy (FLIM) of endogenous nicotinamide adenine dinucleotide (NADH), a key metabolic coenzyme, offers a non-invasive, diagnostic indicator of disease progression, and treatment response. The model-independent phasor analysis approach leverages FLIM to rapidly evaluate cancer metabolism in response to targeted therapy. AIM: We combined lifetime and phasor FLIM analysis to evaluate the influence of human epidermal growth factor receptor 2 (HER2) inhibition, a prevalent cancer biomarker, on both nuclear and cytoplasmic NAD(P)H of two squamous cell carcinoma (SCC) cultures. While better established, the standard lifetime analysis approach is relatively slow and potentially subject to intrinsic fitting errors and model assumptions. Phasor FLIM analysis offers a rapid, model-independent alternative, but the sensitivity of the bound NAD(P)H fraction to growth factor signaling must also be firmly established. APPROACH: Two SCC cultures with low- and high-HER2 expression, were imaged using multiphoton-excited NAD(P)H FLIM, with and without treatment of the HER2 inhibitor AG825. Cells were challenged with mitochondrial inhibition and uncoupling to investigate AG825's impact on the overall metabolic capacity. Phasor FLIM and lifetime fitting analyses were compared within nuclear and cytoplasmic compartments to investigate epigenetic and metabolic impacts of HER2 inhibition. RESULTS: NAD(P)H fluorescence lifetime and bound fraction consistently decreased following HER2 inhibition in both cell lines. High-HER2 SCC74B cells displayed a more significant response than low-HER2 SCC74A in both techniques. HER2 inhibition induced greater changes in nuclear than cytoplasmic compartments, leading to an increase in NAD(P)H intensity and concentration. CONCLUSIONS: The use of both, complementary FLIM analysis techniques together with quantitative fluorescence intensity revealed consistent, quantitative changes in NAD(P)H metabolism associated with inhibition of growth factor signaling in SCC cell lines. HER2 inhibition promoted increased reliance on oxidative phosphorylation in both cell lines.

Difficult airway response team: a novel quality improvement program for managing hospital-wide airway emergencies.

BACKGROUND: Difficult airway cases can quickly become emergencies, increasing the risk of life-threatening complications or death. Emergency airway management outside the operating room is particularly challenging. METHODS: We developed a quality improvement program-the Difficult Airway Response Team (DART)-to improve emergency airway management outside the operating room. DART was implemented by a team of anesthesiologists, otolaryngologists, trauma surgeons, emergency medicine physicians, and risk managers in 2005 at The Johns Hopkins Hospital in Baltimore, Maryland. The DART program had 3 core components: operations, safety, and education. The operations component focused on developing a multidisciplinary difficult airway response team, standardizing the emergency response process, and deploying difficult airway equipment carts throughout the hospital. The safety component focused on real-time monitoring of DART activations and learning from past DART events to continuously improve system-level performance. This objective entailed monitoring the paging system, reporting difficult airway events and DART activations to a Web-based registry, and using in situ simulations to identify and mitigate defects in the emergency airway management process. The educational component included development of a multispecialty difficult airway curriculum encompassing case-based lectures, simulation, and team building/communication to ensure consistency of care. Educational materials were also developed for non-DART staff and patients to inform them about the needs of patients with difficult airways and ensure continuity of care with other providers after discharge. RESULTS: Between July 2008 and June 2013, DART managed 360 adult difficult airway events comprising 8% of all code activations. Predisposing patient factors included body mass index >40, history of head and neck tumor, prior difficult intubation, cervical spine injury, airway edema, airway bleeding, and previous or current tracheostomy. Twenty-three patients (6%) required emergent surgical airways. Sixty-two patients (17%) were stabilized and transported to the operating room for definitive airway management. There were no airway management-related deaths, sentinel events, or malpractice claims in adult patients managed by DART. Five in situ simulations conducted in the first program year improved DART's teamwork, communication, and response times and increased the functionality of the difficult airway carts. Over the 5-year period, we conducted 18 airway courses, through which >200 providers were trained. CONCLUSIONS: DART is a comprehensive program for improving difficult airway management. Future studies will examine the comparative effectiveness of the DART program and evaluate how DART has impacted patient outcomes, operational efficiency, and costs of care.

A novel role for otolaryngologists in the multidisciplinary Difficult Airway Response Team.

OBJECTIVES/HYPOTHESIS: The Difficult Airway Response Team (DART) was implemented in July 2008 to address emergent difficult airway situations. The main objective of this study was to highlight the unique role and skill set that otolaryngologists bring and their impact on patient outcomes. STUDY DESIGN: Retrospective review of prospectively collected data from the hospital's airway registry. METHODS: We collected data on demographics, airway characteristics, airway management techniques used by each specialty, and clinical outcomes (such as cricothyrotomies) for patients for whom a code was activated between July 2006 and June 2010. We compared data between pre- and post-DART cohorts and between DART and non-DART patients using a matched case-control approach. RESULTS: Of the 2,826 codes, 90 patients required DART management between July 2008 and June 2010. Body mass index, cervical spine injury/fixation, history of difficult airway, head and neck mass, and oropharyngeal and/or supraglottic angioedema were identified as significant predictors for DART activation. Forty-nine (60%) patients' airways were secured by anesthesiologists, 30 (36%) by otolaryngologists, and three (4%) by trauma surgeons. Otolaryngologists were able to use specialized techniques such as Holinger and Dedo laryngoscopes to significantly decrease the number of cricothyrotomies from seven (0.73%) pre-DART implementation to four (0.21%) post-DART implementation. CONCLUSIONS: Otolaryngologists were able to decrease the need for cricothyrotomies using specialized techniques for patients with difficult airways. Otolaryngologists bring a special skill set to the DART that is beyond the scope of anesthesiologists and trauma surgeons and that can improve patient outcomes by preventing unnecessary emergency surgical airways.

Multidisciplinary team approach in the management of tracheostomy patients.

OBJECTIVE: To examine whether the implementation of a multidisciplinary percutaneous tracheostomy team decreases complications, improves efficiency in patient care, and reduces length of stay and cost in patients undergoing percutaneous tracheostomy. STUDY DESIGN: Case series with planned data collection. SETTING: Urban, academic, tertiary care medical center. SUBJECTS AND METHODS: Patients who underwent a percutaneous tracheostomy in 2004 and 2008, before and after the formation of a multidisciplinary percutaneous tracheostomy team, were included in the study. Data for the study were retrieved from a tracheostomy database. Measured outcomes include complications, efficiency, length of stay, and cost. RESULTS: Complications such as airway bleeding and physiological disturbances decreased significantly in 2008 as compared with 2004. The percentage of patients who received a tracheostomy within 2 days increased from 42.3% to 92% (2004 vs 2008), showing improvement in efficiency of care. There was no significant difference between the groups in terms of infection rate, length of stay, or mortality. However, in a subanalysis, the length of stay was found to be decreased in patients whose primary diagnosis was a neurological disorder. Finally, despite the necessity of a hospital-based subsidy, the team approach yielded substantial financial benefit to the medical center. CONCLUSIONS: Airway bleeding, physiological disturbances, and efficiency of care improved after the institution of a multidisciplinary percutaneous tracheostomy team approach and may have a favorable impact on health care costs.