Assessment of Automated Flow Cytometry Data Analysis Tools within Cell and Gene Therapy Manufacturing.

Flow cytometry is widely used within the manufacturing of cell and gene therapies to measure and characterise cells. Conventional manual data analysis relies heavily on operator judgement, presenting a major source of variation that can adversely impact the quality and predictive potential of therapies given to patients. Computational tools have the capacity to minimise operator variation and bias in flow cytometry data analysis; however, in many cases, confidence in these technologies has yet to be fully established mirrored by aspects of regulatory concern. Here, we employed synthetic flow cytometry datasets containing controlled population characteristics of separation, and normal/skew distributions to investigate the accuracy and reproducibility of six cell population identification tools, each of which implement different unsupervised clustering algorithms: Flock2, flowMeans, FlowSOM, PhenoGraph, SPADE3 and SWIFT (density-based, k-means, self-organising map, k-nearest neighbour, deterministic k-means, and model-based clustering, respectively). We found that outputs from software analysing the same reference synthetic dataset vary considerably and accuracy deteriorates as the cluster separation index falls below zero. Consequently, as clusters begin to merge, the flowMeans and Flock2 software platforms struggle to identify target clusters more than other platforms. Moreover, the presence of skewed cell populations resulted in poor performance from SWIFT, though FlowSOM, PhenoGraph and SPADE3 were relatively unaffected in comparison. These findings illustrate how novel flow cytometry synthetic datasets can be utilised to validate a range of automated cell identification methods, leading to enhanced confidence in the data quality of automated cell characterisations and enumerations.

Deep Learning-Based Assessment of Brain Connectivity Related to Obstructive Sleep Apnea and Daytime Sleepiness.

PURPOSE: Obstructive sleep apnea (OSA) is associated with altered pairwise connections between brain regions, which might explain cognitive impairment and daytime sleepiness. By adopting a deep learning method, we investigated brain connectivity related to the severity of OSA and daytime sleepiness. PATIENTS AND METHODS: A cross-sectional design applied a deep learning model on structural brain networks obtained from 553 subjects (age, 59.2 ± 7.4 years; men, 35.6%). The model performance was evaluated with the Pearson's correlation coefficient (R) and probability of absolute error less than standard deviation (PAE

Factors Explaining Socio-Economic Inequalities in Survival from Colon Cancer: A Causal Mediation Analysis.

BACKGROUND: Socio-economic inequalities in colon cancer survival exist in high-income countries, but the reasons are unclear. We assessed the mediating effects of stage at diagnosis, comorbidities, and treatment (surgery and intravenous chemotherapy) on survival from colon cancer. METHODS: We identified 2,203 people aged 15 to 79 years with first primary colon cancer diagnosed in Victoria, Australia, between 2008 and 2011. Colon cancer cases were identified through the Victorian Cancer Registry (VCR), and clinical information was obtained from hospital records. Deaths till December 31, 2016 (n = 807), were identified from Victorian and national death registries. Socio-economic disadvantage was based on residential address at diagnosis. For stage III disease, we decomposed its total effect into direct and indirect effects using interventional mediation analysis. RESULTS: Socio-economic inequalities in colon cancer survival were not explained by stage and were greater for men than women. For men with stage III disease, there were 161 [95% confidence interval (CI), 67-256] additional deaths per 1,000 cases in the 5 years following diagnosis for the most disadvantaged compared with the least disadvantaged. The indirect effects through comorbidities and intravenous chemotherapy explained 6 (95% CI, -10-21) and 15 (95% CI, -14-44) per 1,000 of these additional deaths, respectively. Surgery did not explain the observed gap in survival. CONCLUSIONS: Disadvantaged men have lower survival from stage III colon cancer that is only modestly explained by having comorbidities or not receiving chemotherapy after surgery. IMPACT: Future studies should investigate the potential mediating role of factors occurring beyond the first year following diagnosis, such as compliance with surveillance for recurrence and supportive care services.

Science-based assessment of source materials for cell-based medicines: report of a stakeholders workshop.

Human pluripotent stem cells (hPSCs) have the potential to transform medicine. However, hurdles remain to ensure safety for such cellular products. Science-based understanding of the requirements for source materials is required as are appropriate materials. Leaders in hPSC biology, clinical translation, biomanufacturing and regulatory issues were brought together to define requirements for source materials for the production of hPSC-derived therapies and to identify other key issues for the safety of cell therapy products. While the focus of this meeting was on hPSC-derived cell therapies, many of the issues are generic to all cell-based medicines. The intent of this report is to summarize the key issues discussed and record the consensus reached on each of these by the expert delegates.

Modeling focusing Gaussian beams in a turbid medium with Monte Carlo simulations.

Monte Carlo techniques are the gold standard for studying light propagation in turbid media. Traditional Monte Carlo techniques are unable to include wave effects, such as diffraction; thus, these methods are unsuitable for exploring focusing geometries where a significant ballistic component remains at the focal plane. Here, a method is presented for accurately simulating photon propagation at the focal plane, in the context of a traditional Monte Carlo simulation. This is accomplished by propagating ballistic photons along trajectories predicted by Gaussian optics until they undergo an initial scattering event, after which, they are propagated through the medium by a traditional Monte Carlo technique. Solving a known problem by building upon an existing Monte Carlo implementation allows this method to be easily implemented in a wide variety of existing Monte Carlo simulations, greatly improving the accuracy of those models for studying dynamics in a focusing geometry.

Assessment of tissue heating under tunable near-infrared radiation.

The time-temperature effects of laser radiation exposure are investigated as a function of wavelength. Here, we report the thermal response of bulk tissue as a function of wavelength from 700 to 1064 nm. Additionally, Monte Carlo simulations were used to verify the thermal response measured and predict damage thresholds based on the response.

Bright emission from a random Raman laser.

Random lasers are a developing class of light sources that utilize a highly disordered gain medium as opposed to a conventional optical cavity. Although traditional random lasers often have a relatively broad emission spectrum, a random laser that utilizes vibration transitions via Raman scattering allows for an extremely narrow bandwidth, on the order of 10 cm(-1). Here we demonstrate the first experimental evidence of lasing via a Raman interaction in a bulk three-dimensional random medium, with conversion efficiencies on the order of a few percent. Furthermore, Monte Carlo simulations are used to study the complex spatial and temporal dynamics of nonlinear processes in turbid media. In addition to providing a large signal, characteristic of the Raman medium, the random Raman laser offers us an entirely new tool for studying the dynamics of gain in a turbid medium.

Assessment of therapeutic options for mild obstructive sleep apnea using cardiopulmonary coupling measures.

OBJECTIVES: To examine the efficacy of various therapeutic modalities for mild obstructive sleep apnea using cardiopulmonary coupling variables of sleep quality. METHODS: A 67-year-old Caucasian subject's sleep was recorded at home for 10 nights using a type 3 sleep recording device that measured ECG and body position, followed by generation of the cardiopulmonary sleep spectrogram. Three baseline nights, one night with a sleep jacket containing 3 tennis balls to restrict sleep in the supine position, 2 nights with oxygen only delivered via a nasal cannula at a flow rate of 2 L/minute, 2 nights with a mandible advancing appliance (MAA) only, and 2 nights using oxygen at 2 L/minute with the MAA were compared. RESULTS: Baseline sleep quality estimated using the ratio of high-frequency and low-frequency coupling (1.03) was below the expected normal adult values ranging from 1.67-4.0. The sleep quality ratio was significantly higher (2.08) using the MAA alone compared to baseline, sleep position restriction (1.61), oxygen therapy (0.81), and the combination of MAA with oxygen (1.66). CONCLUSION: Sleep quality measured objectively using cardiopulmonary coupling variables differentiated the efficacy of therapeutic options for mild obstructive sleep apnea. Such an approach may have practical utility.

Optical properties of ocular tissues in the near infrared region.

Near infrared characterization of optical properties of aqueous humor and vitreous humor of healthy human and bovine eyes has been performed. The indices of refraction (n) of these ocular tissues were determined using a Michelson interferometer. The total diffuse reflection (R (d)) and total transmission (T (t)) measurements had been taken for individual ocular tissue by using a double-integrating sphere setup and infrared laser diodes. The inverse adding doubling (IAD) computational method based on the diffusion approximation and radiative transport theory was applied to the measured values of n, R (d), and T (t) to calculate the optical absorption and scattering coefficients of the human and bovine ocular tissues. The scattering anisotropy value was determined by iteratively running the IAD method program and a Monte Carlo simulation of light-tissue interaction until the minimum difference in experimental and computed value for T (t) was realized. A comparison between the optical characterization of human and bovine ocular samples was also made.

Adults with ADHD and sleep complaints: a pilot study identifying sleep-disordered breathing using polysomnography and sleep quality assessment.

OBJECTIVE: ADHD and sleep-disordered breathing are both prevalent in adulthood. Because both conditions may be responsible for similar symptoms of cognitive impairment, the authors investigate whether their presentation may overlap in adults diagnosed with ADHD. METHOD: Data are collected from six adults with sleep complaints who were diagnosed with ADHD using rigorous clinical criteria. All participants undergo overnight polysomnography and complete questionnaires about sleep quality, circadian sleep pattern, and daytime fatigue. RESULTS: On standardized measures, all participants report poor sleep quality, two report daytime fatigue, and none report distinct deviation from normal sleep and wake cycle pattern. Polysomnography reveals evidence of sleep-disordered breathing and sleep fragmentation in all participants. CONCLUSION: Objective evidence of breathing-related sleep disorders can be found in some adults with carefully diagnosed ADHD who report sleep complaints. This report highlights the importance of identifying treatable sleep disorder comorbidity in adults with ADHD.