Assessment of cell cycle progression and mitotic slippage by videomicroscopy.

Senescence is a state of irreversible cell cycle arrest accompanied by the acquisition of the senescence-associated secretory phenotype (SASP), which is activated in response to a variety of damaging stimuli, including genotoxic therapy. Accumulating evidence indicates that mitotic stress also promotes entry into senescence. This occurs via a mechanism involving defective mitoses and mitotic arrest, followed by abortion of cell division and slippage in the G1 phase. In this process, mitotic slippage leads to the generation of senescent cells characterized by a large cell body and a multinucleated and/or enlarged nuclear size. Here, we provide a detailed protocol for the assessment of cell proliferation and mitotic slippage in colorectal cancer cells upon pharmacological inhibition of the mitotic kinesin KIF11, best known as EG5. This approach can be used for preliminary characterization of senescence induction by therapeutics, but requires validation with standard senescence assays.

Guideline for in vivo assessment of adherent and rolling leukocytes in human skin microvasculature via reflectance confocal videomicroscopy.

OBJECTIVE: To develop a guideline that reliably identifies cutaneous adherent and rolling leukocytes from mimicking scenarios via in vivo reflectance confocal videomicroscopy. METHODS: We used a clinical reflectance confocal microscope, the VivaScope 1500, to acquire 1522 videos of the upper dermal microcirculation from 12 healthy subjects and 60 patients after allogeneic hematopoietic cell transplantation. Blinded to clinical information, two trained raters independently counted the number of adherent and rolling leukocytes in 88 videos. Based on discrepancies in the initial assessments, we developed a guideline to identify both types of leukocyte-endothelial interactions via a modified Delphi method (without anonymity). To test the guideline's ability to improve the inter-rater reliability, the two raters assessed the remaining 1434 videos by using the guideline. RESULTS: We demonstrate a guideline that consists of definitions, a step-by-step flowchart, and corresponding visuals of adherent and rolling leukocytes and mimicking scenarios. The guideline improved the inter-rater reliability of the manual assessment of both interactions. The intraclass correlation coefficient (ICC) of adherent leukocyte counts increased from 0.056 (95% confidence interval: 0-0.236, n = 88 videos, N = 10 subjects) to 0.791 (0.770-0.809, n = 1434, N = 67). The ICC of rolling leukocyte counts increased from 0.385 (0.191-0.550, n = 88, N = 10) to 0.626 (0.593-0.657, n = 1434, N = 67). Intra-rater ICC post-guideline was 0.953 (0.886-0.981, n = 20, N = 12) and 0.956 (0.894-0.983, n = 20, N = 12) for adherent and rolling, respectively. CONCLUSION: The guideline aids in the manual identification of adherent and rolling leukocytes via in vivo reflectance confocal videomicroscopy.

Assessment of dispersion of airborne particles of oral/nasal fluid by high flow nasal cannula therapy.

BACKGROUND: Nasal High Flow (NHF) therapy delivers flows of heated humidified gases up to 60 LPM (litres per minute) via a nasal cannula. Particles of oral/nasal fluid released by patients undergoing NHF therapy may pose a cross-infection risk, which is a potential concern for treating COVID-19 patients. METHODS: Liquid particles within the exhaled breath of healthy participants were measured with two protocols: (1) high speed camera imaging and counting exhaled particles under high magnification (6 participants) and (2) measuring the deposition of a chemical marker (riboflavin-5-monophosphate) at a distance of 100 and 500 mm on filter papers through which air was drawn (10 participants). The filter papers were assayed with HPLC. Breathing conditions tested included quiet (resting) breathing and vigorous breathing (which here means nasal snorting, voluntary coughing and voluntary sneezing). Unsupported (natural) breathing and NHF at 30 and 60 LPM were compared. RESULTS: Imaging: During quiet breathing, no particles were recorded with unsupported breathing or 30 LPM NHF (detection limit for single particles 33 µm). Particles were detected from 2 of 6 participants at 60 LPM quiet breathing at approximately 10% of the rate caused by unsupported vigorous breathing. Unsupported vigorous breathing released the greatest numbers of particles. Vigorous breathing with NHF at 60 LPM, released half the number of particles compared to vigorous breathing without NHF.Chemical marker tests: No oral/nasal fluid was detected in quiet breathing without NHF (detection limit 0.28 µL/m3). In quiet breathing with NHF at 60 LPM, small quantities were detected in 4 out of 29 quiet breathing tests, not exceeding 17 µL/m3. Vigorous breathing released 200-1000 times more fluid than the quiet breathing with NHF. The quantities detected in vigorous breathing were similar whether using NHF or not. CONCLUSION: During quiet breathing, 60 LPM NHF therapy may cause oral/nasal fluid to be released as particles, at levels of tens of µL per cubic metre of air. Vigorous breathing (snort, cough or sneeze) releases 200 to 1000 times more oral/nasal fluid than quiet breathing (p < 0.001 with both imaging and chemical marker methods). During vigorous breathing, 60 LPM NHF therapy caused no statistically significant difference in the quantity of oral/nasal fluid released compared to unsupported breathing. NHF use does not increase the risk of dispersing infectious aerosols above the risk of unsupported vigorous breathing. Standard infection prevention and control measures should apply when dealing with a patient who has an acute respiratory infection, independent of which, if any, respiratory support is being used. CLINICAL TRIAL REGISTRATION: ACTRN12614000924651.

Noninvasive, in vivo assessment of the cervical microcirculation using incident dark field imaging.

AIM: This study evaluates the feasibility of handheld vital microscopy for noninvasive, objective assessment of the microcirculation of the human uterine cervix. We qualitatively and quantitatively describe the microcirculation in healthy subjects in order to provide a basis for its application in cervical pathology. METHODS: Incident dark field imaging was used to image the microcirculation in four quadrants of the uterine ectocervix in ten healthy participants. If the squamocolumnar junction was visible, measurements were repeated on the endocervical columnar epithelium as well. Image acquisition time was recorded and participants scored the experienced level of discomfort. Angioarchitecture was classified according to Weber's classification. Quantitative parameters included capillary density (CD), total and perfused vessel density (TVD, PVD), proportion of perfused vessels (PPV) and microvascular flow index (MFI). RESULTS: Image acquisition was easy, fast and well tolerated. Angioarchitecture was characterized by two distinctive and organized patterns; capillary loops underneath the squamous epithelium of the ectocervix and vascular networks underneath the columnar epithelium. In the image sequences containing capillary loops, mean CD was 33.2 cpll/mm2 (95% CI 28.2-38.2 cpll/mm2). In the image sequences with vascular networks, mean TVD was 12.5 mm/mm2 (95% CI 11.2-13.77 mm/mm2), mean PVD was 12.2 (95% CI 11.0-13.5 mm/mm2), MFI was 3 and PPV was 100%. CONCLUSIONS: Incident dark field imaging allows for noninvasive, real time visualization and objective evaluation and quantification of the microcirculation of the uterine cervix. The organized vascular patterns and optimal perfusion observed in healthy subjects allow for comparison with cervical pathology, for example in patients with cervical dysplasia or cervical cancer.

Probe-based Confocal Laser Endomicroscopy for In Vivo Assessment of Histological Healing in Ulcerative Colitis: Development and Validation of the ENHANCE Index.

BACKGROUND AND AIMS: Histological healing may represent the ultimate therapeutic goal in ulcerative colitis [UC], but it requires biopsies. Our aim was to develop a non-invasive index able to assess histological disease activity in ulcerative colitis, using probe-based confocal laser endomicroscopy [pCLE]. METHODS: One hundred patients with quiescent UC were prospectively included in five French centres. After fluorescein intravenous injection, during colonoscopy, the colorectal mucosa was analysed by white light imaging and pCLE, and then biopsied in different locations. Five endoscopists performed central reading of pCLE images blinded to clinical, endoscopic, and histological data. One expert pathologist performed a central histological reading [Nancy index: gold standard]. Univariate and multivariate analyses were performed to identify the endomicroscopic items associated with the presence of histologically active disease. RESULTS: Over 1000 pCLE videos sequences performed in 100 UC patients in endoscopic remission [Mayo 0 and 1] were evaluated. We observed that vessel diameter >20 µm, dilated crypt lumen, fluorescein leakage, and irregular crypt architecture were statistically associated with histologically proven inflammation according to the Nancy index. Hence, we built a pCLE index of mucosal inflammation with overall accuracy of 79.6% and overall sensitivity and specificity of, respectively, 57.8% and 82.8%. Negative predictive value, especially when a pCLE index ≤1 was observed, was high [93.1%]. CONCLUSIONS: Using a robust methodology, large vessel diameter, dilated crypt lumen, fluorescein leakage,and irregular crypt architecture are reliable endomicroscopic items defining the ENHANCE index for real-time assessment of histological disease activity in UC.

Cardiac function and blood flow hemodynamics assessment of zebrafish (Danio rerio) using high-speed video microscopy.

BACKGROUND: In the last few decades, zebrafish (Danio rerio) were introduced as a model organism to investigate human diseases including cardiovascular and neuronal disorders. In most zebrafish investigations, cardiac function and blood flow hemodynamics need to be assessed to study the effects of the interference on the cardiovascular system. For heart function assessment, most important parameters include heart rate, cardiac output, ejection fraction, fractional area change, and fractional shortening. METHODS: A 10 s high-speed video of beating heart and flowing blood within major vessels of zebrafish that are less than 5 days post fertilization (dpf) were recorded via a stereo microscope equipped with a high speed camera. The videos were analyzed using MicroZebraLab and image J software for the assessment of cardiac function. RESULTS: Using the technique described here, we were able to simply yet effectively assess cardiac function and blood flow dynamics of normal zebrafish embryos. We believe that the practical method presented here will help cardiac researchers using the zebrafish as a model to examine cardiac function by using tools that could be available in their laboratory.

Cardiac safety assessment with motion field imaging analysis of human iPS cell-derived cardiomyocytes is improved by an integrated evaluation with cardiac ion channel profiling.

We validated a motion field imaging (MFI) assay with human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) as a model to assess multiple cardiac liabilities by comparing the guinea-pig Langendorff heart with hiPS-CMs using 4 reference compounds and 9 internal compounds. We investigated repolarization duration, beating rate (BR), conduction speed, contractility, and inhibitory profile of three cardiac ion channels: hERG, Cav1.2, and Nav1.5. For repolarization, the contraction-relaxation duration (CRDc) of hiPS-CMs was generally consistent with the QTc interval of Langendorff heart. However, 2 internal compounds shortened CRDc despite QTc prolongation in Langendorff heart. Cardiac ion channel profiling revealed that hiPS-CMs could not be used to detect QTc prolongation when the value of Cav1.2 IC50 / hERG IC50 for a compound was between 1 and 10, whereas hiPS-CMs showed responses largely consistent with Langendorff heart when Cav1.2 IC50 / hERG IC50 was below 1 or above 10. The accuracy of hiPS-CMs for the BR was not high, mainly because the BR of hiPS-CMs was increased by an inhibition of Cav1.2. The hiPS-CMs were highly sensitive to conduction speed and contractility, able to detect QRS widening caused by Nav1.5-inhibition, as well as decreased LVdP/dtmax caused by the inhibition of Cav1.2 and/or Nav1.5. In conclusion, the MFI assay with hiPS-CMs would be useful for evaluating multiple cardiac liabilities. The ion channel profile helps to interpret the results of MFI assay and correctly evaluate cardiac risks. Therefore, an integrated cardiac safety assessment with MFI and ion channel profiling is recommended.

Sidestream Dark Field (SDF) imaging of oral microcirculation in the assessment of systemic sclerosis.

INTRODUCTION: Systemic sclerosis (SSc) is a systemic disease characterised by abnormalities in small blood vessels, skin and organ fibrosis. It is assessed using generalised skin thickening scores, autoantibodies and nailfold capillaroscopy. Sidestream Dark Field imaging (SDF) is a non-invasive imaging tool that assesses microcirculation. This study aims to investigate the potential of using SDF as a diagnostic tool in SSc. METHOD: Oral microcirculation of 20 patients with SSc was compared to 20 age and gender matched controls using SDF imaging. Sublingual, buccal and incisor regions of the mouth were examined. All volunteers were female averaging 48.0 (24-64) years old. Vasculature was assessed by calculating the De Backer score and Functional Capillary Density (FCD) on an imaging software. RESULTS: At all regions of the mouth, SSc patients had a significantly lower De Backer score compared to controls (SSc 3.484 ±â€¯0.1361/mm vs Control 5.184 ±â€¯0.1896/mm, unpaired t-test p < 0.0001). The SSc patients showed significantly lower FCDs compared to controls at all areas as well (SSc 19.65 ±â€¯0.9445% vs Control 29.45 ±â€¯1.681%, unpaired t-test, p < 0.0001). The incisor regions had significantly higher De Backer and FCD scores than buccal and sublingual regions in both control and SSc patients (one way anova, p < 0.05). De Backer/FCD scores showed significant correlation against Rodnan Skin Scores in patients with SSc (Pearson correlation, p < 0.05). CONCLUSIONS: SSc patients showed decreased oral vasculature compared to controls. SDF imaging has shown the ability to be a useful diagnostic tool in the assessment of SSc.

Patient Attitude towards Videodermatoscopy for the Detection of Skin Cancer: A Cross-Sectional Study.

BACKGROUND: Videodermatoscopy (VD) is a useful device for supporting dermatologists in the distinction between benign and malignant lesions. However, only few patients have access to VD in daily practice. OBJECTIVES: To investigate patient attitudes towards VD. METHOD: A cross-sectional study was conducted between May and June 2018. Patients were asked to complete a self-administered questionnaire on the popularity of VD. Descriptive analysis was performed including contingency tables and χ2 tests to investigate associations between sociodemographic data and the popularity of VD. RESULTS: A total of 61.2% (123/201) of the patients had not heard of VD at the time of assessment or were unsure. Of the 38.8% of patients (78/201) who already knew of VD, 64.1% (50/78) reported that they had already been investigated by VD; 57.5% (111/193) were willing to pay an extra fee for VD. A high level of education and private insurance status had a statistically significant association with the popularity of VD (p = 0.036 and p = 0.026, respectively). CONCLUSIONS: There was a strong information deficit, especially in patients with lower education and statutory health insurance. Nevertheless, the willingness to pay an extra fee for a VD-assisted skin examination was high. Dermatologists should actively offer and inform their patients about VD when performing skin cancer screening.

Low-Cost Stereoscopic Recordings of Neurologic Surgery Operative Microscopy for Anatomic Laboratory Training.

OBJECTIVE: Stereoscopic video recordings of operative microscopy during neuroanatomic dissections are an important component of surgical training and research in well-financed medical schools and teaching hospitals. However, the high cost of the latest operative microscopes with integrated video recording equipment can be a limiting factor in their worldwide use. The aim of the present work is to provide a simple low-cost 3-dimensional (3D) stereoscopic operative microscope recording system that can be used even in economically and resource-limited locations. This is achieved by using readily available smartphones, smartphone accessories, and computer software. METHODS: Stereoscopic recording is accomplished by attaching and aligning matched or similar smartphones to the eyepieces of an operative microscope using readily available smartphone mounting connectors. Video recordings from the smartphones are then transferred to a personal computer and processed with a video-editing software to generate stereoscopic movies that are viewed on a smartphone using virtual-reality glasses. RESULTS: The setup time to mount and align the smartphone cameras typically requires 15-30 minutes. Video image quality and 3D depth presentation is more than sufficient for surgical training and research purposes. The implementation cost ranges from $1,315-$7,066, or much less if smartphones and a computer are already available. CONCLUSIONS: The 3D video system demonstrated herein can be implemented on any type of operative microscope, including older units for which commercial stereo recording systems are not available. The system and method presented herein can be readily and affordably implemented in low-budget environments for clinical training and research.

A systematic review on the quality of measurement techniques for the assessment of burn wound depth or healing potential.

PURPOSE: Reliable and valid assessment of burn wound depth or healing potential is essential to treatment decision-making, to provide a prognosis, and to compare studies evaluating different treatment modalities. The aim of this review was to critically appraise, compare and summarize the quality of relevant measurement properties of techniques that aim to assess burn wound depth or healing potential. METHODS: A systematic literature search was performed using PubMed, EMBASE and Cochrane Library. Two reviewers independently evaluated the methodological quality of included articles using an adapted version of the Consensus-based Standards for the selection of health Measurement INstruments (COSMIN) checklist. A synthesis of evidence was performed to rate the measurement properties for each technique and to draw an overall conclusion on quality of the techniques. RESULTS: Thirty-six articles were included, evaluating various techniques, classified as (1) laser Doppler techniques; (2) thermography or thermal imaging; (3) other measurement techniques. Strong evidence was found for adequate construct validity of laser Doppler imaging (LDI). Moderate evidence was found for adequate construct validity of thermography, videomicroscopy, and spatial frequency domain imaging (SFDI). Only two studies reported on the measurement property reliability. Furthermore, considerable variation was observed among comparator instruments. CONCLUSIONS: Considering the evidence available, it appears that LDI is currently the most favorable technique; thereby assessing burn wound healing potential. Additional research is needed into thermography, videomicroscopy, and SFDI to evaluate their full potential. Future studies should focus on reliability and measurement error, and provide a precise description of which construct is aimed to measure.

In vivo assessment of the human cerebral microcirculation and its glycocalyx: A technical report.

INTRODUCTION: The cerebral microcirculation and its glycocalyx, a matrix coating the luminal endothelium, are key regulators of capillary permeability and cerebral blood flow. Microvascular abnormalities are described in several neurological disorders. However, assessment of the cerebral microcirculation and glycocalyx has mainly been performed ex vivo. NEW METHOD: Here, the technical feasibility of in vivo assessment of the human cerebral microcirculation and its glycocalyx using sidestream dark field (SDF) imaging is discussed. Intraoperative assessment requires the application of a sterile drape covering the camera (slipcover). First, sublingual measurements with and without slipcover were performed in a healthy control to assess the impact of this slipcover. Subsequently, using SDF imaging, the sublingual (reference), cortical, and hippocampal microcirculation and glycocalyx were evaluated in patients who underwent resective brain surgery as treatment for drug-resistant temporal lobe epilepsy. Finally, vessel density, and the perfused boundary region (PBR), a validated gauge of glycocalyx health, were calculated using GlycoCheck© software. RESULTS: The addition of a slipcover affects vessel density and PBR values in a control subject. The cerebral measurements in five patients were more difficult to obtain than the sublingual ones. This was probably at least partly due to the introduction of a sterile slipcover. Results on vessel density and PBR showed similar patterns at all three measurement sites. COMPARISON WITH EXISTING METHODS: This is the first report on in vivo assessment of the human cerebrovascular glycocalyx. Assessment of the glycocalyx is an additional application of in vivo imaging of the cerebral microcirculation using SDF technique. This method enables functional analysis of the microcirculation and glycocalyx, however the addition of a sterile slipcover affects the measurements. CONCLUSIONS: SDF imaging is a safe, quick, and straightforward technique to evaluate the functional cerebral microcirculation and glycocalyx. Because of their eminent role in cerebral homeostasis, this method may significantly add to research on the role of vascular pathophysiology underling various neurological disorders.

Assessment of Human Adipose Tissue Microvascular Function Using Videomicroscopy.

While obesity is closely linked to the development of metabolic and cardiovascular disease, little is known about mechanisms that govern these processes. It is hypothesized that pro-atherogenic mediators released from fat tissues particularly in association with central/visceral adiposity may promote pathogenic vascular changes locally and systemically, and the notion that cardiovascular disease may be the consequence of adipose tissue dysfunction continues to evolve. Here, we describe a unique method of videomicroscopy that involves analysis of vasodilator and vasoconstrictor responses of intact small human arterioles removed from the adipose depot of living human subjects. Videomicroscopy is used to examine functional properties of isolated microvessels in response to pharmacological or physiological stimuli using a pressured system that mimics in vivo conditions. The technique is a useful approach to gain understanding of the pathophysiology and molecular mechanisms that contribute to vascular dysfunction locally within the adipose tissue milieu. Moreover, abnormalities in the adipose tissue microvasculature have also been linked with systemic diseases. We applied this technique to examine depot-specific vascular responses in obese subjects. We assessed endothelium-dependent vasodilation to both increased flow and acetylcholine in adipose arterioles (50 - 350 µm internal diameter, 2 - 3 mm in length) isolated from two different adipose depots during bariatric surgery from the same individual. We demonstrated that arterioles from visceral fat exhibit impaired endothelium-dependent vasodilation compared to vessels isolated from the subcutaneous depot. The findings suggest that the visceral microenvironment is associated with vascular endothelial dysfunction which may be relevant to clinical observation linking increased visceral adiposity to systemic disease mechanisms. The videomicroscopy technique can be used to examine vascular phenotypes from different fat depots as well as compare findings across individuals with different degrees of obesity and metabolic dysfunction. The method can also be used to examine vascular responses longitudinally in response to clinical interventions.

Time-lapse imaging of cleavage divisions in embryo quality assessment.

In vitro fertilization (IVF) is one of the most important procedures for treating infertility. As several embryos are usually produced in a single IVF cycle, it is crucial to select only the most viable ones for transfer to the patient. Morphokinetics, i.e. analysis of the dynamics of cleavage divisions and processes such as compaction and cavitation, has provided both biologists and clinicians with a new set of data regarding embryonic behaviour during preimplantation development and its association with embryo quality. In the current review, we focus on biological significance of morphokinetic parameters and show how they can be used to predict a reproductive outcome. We also explain the statistics behind the predictive algorithms and discuss the future perspectives of morphokinetics.

Objective assessment of acne.

A precise and reliable assessment of acne severity is unarguably the most essential clinical method when it comes to monitoring and choosing optimal treatment in the daily practice. Since the early 1960s, different severity assessment systems have been described in the literature. The two commonly used concepts are global gradings and lesion counting. Both systems have been controversially discussed as to which is more reliable and providing an objective outcome measurement tool; however, both have some subjectivity involved. More objective methods for assessing the severity of acne vulgaris include photography, fluorescence photography, polarized light photography, video microscopy, and multispectral imaging. Such techniques have limitations such as high cost, complex and sophisticated apparatus, and a sometimes time-consuming imaging process. There are newly developed technologies that could avoid the problems of inter- and intrarater subjectivity.

Protocol for intraoperative assessment of the human cerebrovascular glycocalyx.

INTRODUCTION: Adequate functioning of the blood-brain barrier (BBB) is important for brain homoeostasis and normal neuronal function. Disruption of the BBB has been described in several neurological diseases. Recent reports suggest that an increased permeability of the BBB also contributes to increased seizure susceptibility in patients with epilepsy. The endothelial glycocalyx is coating the luminal side of the endothelium and can be considered as the first barrier of the BBB. We hypothesise that an altered glycocalyx thickness plays a role in the aetiology of temporal lobe epilepsy (TLE), the most common type of epilepsy. Here, we propose a protocol that allows intraoperative assessment of the cerebrovascular glycocalyx thickness in patients with TLE and assess whether its thickness is decreased in patients with TLE when compared with controls. METHODS AND ANALYSIS: This protocol is designed as a prospective observational case-control study in patients who undergo resective brain surgery as treatment for TLE. Control subjects are patients without a history of epileptic seizures, who undergo a craniotomy or burr hole surgery for other indications. Intraoperative glycocalyx thickness measurements of sublingual, cortical and hippocampal microcirculation are performed by video microscopy using sidestream dark-field imaging. Demographic details, seizure characteristics, epilepsy risk factors, intraoperative haemodynamic parameters and histopathological evaluation are additionally recorded. ETHICS AND DISSEMINATION: This protocol has been ethically approved by the local medical ethical committee (ID: NL51594.068.14) and complies with the Declaration of Helsinki and principles of Good Clinical Practice. Informed consent is obtained before study enrolment and only coded data will be stored in a secured database, enabling an audit trail. Results will be submitted to international peer-reviewed journals and presented at international conferences. TRIAL REGISTRATION NUMBER: NTR5568.

Time-lapse morphokinetic assessment has low to moderate ability to predict euploidy when patient- and ovarian stimulation-related factors are taken into account with the use of clustered data analysis.

OBJECTIVE: To study whether time-lapse morphokinetic (TLM) assessment predicts ploidy status when patient- and ovarian stimulation-related factors are taken into account. DESIGN: Retrospective cohort study. SETTING: Private IVF clinic. PATIENT(S): In total, 103 consecutive patients (415 blastocysts) were included. All embryos were individually cultured in a time-lapse incubator from intracytoplasmic sperm injection up to trophectoderm biopsy. Following trophectoderm biopsy on day 5 or 6, blastocysts were vitrified and 23 TLM parameters were analyzed. INTERVENTION(S): Correlations between patient- and ovarian stimulation-related factors and TLM parameters were tested in a multilevel mixed-effects linear regression model and assessed by means of intraclass correlation coefficient (ICC). MAIN OUTCOME MEASURE(S): Predictive ability of TLM parameters for euploidy. RESULT(S): The majority of TLM parameters had ICCs of 16%-47%. None of the patient- or ovarian stimulation-related factor had any systematic effect on any TLM parameter; however, body mass, total FSH dose, duration of infertility, number of previous cycles, antral follicle count, ovarian stimulation protocol, and E2 on the trigger day had a significant impact on some TLM parameters. With the use of multilevel mixed-effects logistic regression analysis, of the ten TLM parameters that were initially noted to be significantly different among euploid and aneuploid blastocysts in the univariate analysis, only five remained significant. However, the areas under the receiver operating characteristic curves at regression analysis were low, ranging from 0.55 to 0.63. CONCLUSION(S): Five TLM parameters, all related to timing of blastocyst development, have limited ability to predict euploidy when patient- and ovarian stimulation-related factors are taken into account.

Safety and feasibility of sublingual microcirculation assessment in the emergency department for civilian and military patients with traumatic haemorrhagic shock: a prospective cohort study.

OBJECTIVES: Sublingual microcirculatory monitoring for traumatic haemorrhagic shock (THS) may predict clinical outcomes better than traditional blood pressure and cardiac output, but is not usually performed until the patient reaches the intensive care unit (ICU), missing earlier data of potential importance. This pilot study assessed for the first time the feasibility and safety of sublingual video-microscopy for THS in the emergency department (ED), and whether it yields useable data for analysis. SETTING: A safety and feasibility assessment was undertaken as part of the prospective observational MICROSHOCK study; sublingual video-microscopy was performed at the UK-led Role 3 medical facility at Camp Bastion, Afghanistan, and in the ED in 3 UK Major Trauma Centres. PARTICIPANTS: There were 15 casualties (2 military, 13 civilian) who presented with traumatic haemorrhagic shock with a median injury severity score of 26. The median age was 41; the majority (n=12) were male. The most common injury mechanism was road traffic accident. PRIMARY AND SECONDARY OUTCOME MEASURES: Safety and feasibility were the primary outcomes, as measured by lack of adverse events or clinical interruptions, and successful acquisition and storage of data. The secondary outcome was the quality of acquired video clips according to validated criteria, in order to determine whether useful data could be obtained in this emergency context. RESULTS: Video-microscopy was successfully performed and stored for analysis for all patients, yielding 161 video clips. There were no adverse events or episodes where clinical management was affected or interrupted. There were 104 (64.6%) video clips from 14 patients of sufficient quality for analysis. CONCLUSIONS: Early sublingual microcirculatory monitoring in the ED for patients with THS is safe and feasible, even in a deployed military setting, and yields videos of satisfactory quality in a high proportion of cases. Further investigations of early microcirculatory behaviour in this context are warranted. TRIAL REGISTRATION NUMBER: NCT02111109.

Real-time point of care microcirculatory assessment of shock: design, rationale and application of the point of care microcirculation (POEM) tool.

BACKGROUND: Despite over a decade of research and technological advances, sublingual microcirculatory monitoring has not yet reached clinical utility. Offline analysis is time consuming and occurs away from the patient. A system to assess the microcirculation at the point of care is desirable. We present a novel 5-point grading system (the point of care microcirculation (POEM) scoring system) that can be used at the point of care during non-invasive sublingual microcirculatory monitoring. METHODS: The POEM score is an ordinal scale from 1 (worst) to 5 (best), based on a composite assessment of flow and heterogeneity of four individual sublingual video-microscopy clips. Thirty-two healthcare professionals were trained in how to assign POEM scores. Following training they assigned scores to five test sequences (each consisting of four video clips). They were blinded to clinical status. Inter-user consistency and agreement were assessed using intra-class correlation coefficient (ICC) analysis. In addition, blinded expert scores for 68 video clips were compared to offline computer analysis using traditional microcirculatory parameters including total vessel density (TVD), perfused vessel density (PVD), proportion of perfused vessels (PPV), microcirculatory flow index (MFI) and microcirculatory heterogeneity index (MHI). The time taken to assign each was recorded. RESULTS: Participants showed good inter-rater consistency (ICC 0.83, 95 % CI 0.626, 0.976) and agreement (ICC 0.815, 95 % CI 0.602, 0.974) for assigned POEM scores. Expert scoring of videos correlated with offline values for PVD (R 2 = 0.39; p < 0.05), PPV (R 2 = 0.71; p < 0.001), MFI (R 2 = 0.75; p < 0.001), and MHI (R 2 = 0.68; p < 0.001). POEM scores took less time to assign than conventional offline computer analysis (2 minutes versus 44 minutes). CONCLUSION: We present for the first time a novel 5-point ordinal scale of microcirculatory flow and heterogeneity that can be used at the point of care. It has minimal inter-user variability amongst healthcare professionals after just 1 hour of training. POEM scores take a short time to assign, and correspond well to traditional offline computer-analyzed parameters.

Evaluación de la microcirculación con sidestream dark field (SDF) Microscan® como método de monitorización en microcirugía en modelo porcino / Microcirculation assessment with sidestream dark field (SDF) Microscan® as flap monitoring method in microsurgery in a porcine model

Objetivo: Determinar la factibilidad de la monitorización en microcirugía por medio de la evaluación no invasiva de la microcirculación con sidestream dark field (SDF) y compararla con otros métodos. Materiales y métodos: Estudio experimental. En 8 cerdos se elevó colgajo pectoral y se disecó pedículo. Se llevó a cabo una instalación sucesiva de dispositivos cutáneos para la evaluación de la microcirculación: SDF para evaluar flujo, y near infrared spectroscopy (NIRS) para evaluar saturación de O2 (SatO2). Posteriormente se evaluó la oclusión venosa, arterial y total con pinzamiento durante 180 s. Resultados: SDF en oclusión venosa: disminución del flujo: 51 s (59-62); SDF en oclusión arterial: disminución del flujo: 3 s (1-5); SDF en oclusión vascular total: disminución del flujo: 3,5 s (2-5). NIRS en oclusión venosa: disminución de la SatO2:15,2 ± 5,3%; NIRS en oclusión arterial: disminución de la SatO2 23,9 ± 13,8%; NIRS en oclusión vascular total: disminución de la SatO2 23,85 ± 13,9%. Doppler en oclusión venosa: no desapareció; Doppler en oclusión arterial y oclusión vascular total: desapareció a los 2 s. En cada una de las mediciones, los cambios clínicos fueron más tardíos que los observados con SDF. Conclusión: Es factible la monitorización en microcirugía por medio de la evaluación de la microcirculación con Microscan®. Este método permite realizar el diagnóstico de oclusión vascular más tempranamente que con NIRS y evaluación clínica.

Aim: Determine the feasibility of using SDF Microscan® as a non-invasive method for monitoring free flap microcirculation, and compare it to other methods. Materials and methods: Experimental study. In 8 pigs a pectoral myocutaneous flap was raised. Microcirculation was evaluated using: SDF Microscan®, near infrared spectroscopy (NIRS), clinical examination and Doppler. Venous, arterial and total occlusion was performed by clamping the vascular pedicle. Mean time to blood flow impairment diagnosis was measured. Results: SDF in venous occlusion: reduced microcirculatory flow index at: 51 s (59-62). SDF in arterial occlusion: reduced microcirculatory flow index at: 3 s (1-5). SDF in total vascular occlusion: reduced microcirculatory flow index at: 3.5 s (2-5). NIRS in venous occlusion: SatO2 decrease was 15.2 ± 5.3%. NIRS in arterial occlusion: SatO2 decrease was 23.9 ± 13.8%. NIRS in total vascular occlusion: SatO2 decrease was 23.85 ± 13.9%. Doppler in venous occlusion: The signal did not disappear. Doppler arterial and total vascular occlusion disappears at 2 s. The clinical changes were later than SDF. Conclusion: Microcirculation monitoring is feasible using SDF Microscan® in a pig model. This method allows to detect blood flow disruption earlier than NIRS and clinical evaluation.