A high-throughput pipeline for scalable kit-free RNA extraction

An overreliance on commercial, kit-based RNA extraction in the molecular diagnoses of infectious disease presents a challenge in the event of supply chain disruptions and can potentially hinder testing capacity in times of need. In this study, we adapted a well-established, robust TRIzol-based RNA extraction protocol into a high-throughput format through miniaturization and automation. The workflow was validated by RT-qPCR assay for SARS-CoV-2 detection to illustrate its scalability without interference to downstream diagnostic sensitivity and accuracy. This semi-automated, kit-free approach offers a versatile alternative to prevailing integrated solid-phase RNA extraction proprietary systems, with the added advantage of improved cost-effectiveness for high volume acquisition of quality RNA whether for use in clinical diagnoses or for diverse molecular applications.

Prenatal Screening for Developmental Displacement of the Hip: The BUDDHA (Pre-Birth Ultrasound for Developmental Displacement of the Hip Assessment) Study

Background: developmental dysplasia of the hip has an incidence of 3–5 out of 1000 children. Currently, only postnatal screening is available. Objective: to test the feasibility of a method based on Graf technique application at antenatal ultrasound in assessing the normal development of the hip in unselected term fetuses. Methods: a prospective cohort study in a single university tertiary hospital from January 2017 to January 2020. Single uncomplicated term pregnancies (37–40 weeks) attending our center for routine ultrasound were consecutively recruited for the purpose of the study. A 3D volume acquisition was launched on the coxofemoral joint of the fetus by a single expert operator, and offline analysis was then performed in the multiplanar mode by two operators (blinded to each other analysis) in order to measure the alpha and beta angles according to our modified Graf technique. Intra- and inter-observer variations were calculated. Reference charts for normal values of both angles were produced. Postnatal ultrasound was then performed to measure the Graf angles in newborns, confirming a normal development of the hip. Results: in the study period, 433 uncomplicated term pregnancies underwent 3D ultrasound for the assessment of the fetal hip. One case was subsequently excluded because of confirmed postnatal diagnosis of developmental dysplasia of the hip. The measurement of our modified Graf angles was feasible at prenatal ultrasound with a good reproducibility. The inter-rater and intra-rater reliability of both angles was substantial. Reference charts for normal values of both angles were produced. Conclusions: the evaluation of the coxofemoral joint in fetuses at term of gestation has never been attempted before. The Graf technique application, currently employed at postnatal ultrasound, may also be adapted to prenatal ultrasound with a substantial reproducibility. However, there was no evidence of a linear relationship between prenatal and postnatal alpha angles and beta angles. Further research is needed to establish if developmental dysplasia of the hip could be diagnosed antenatally.

CLEMSite, a software for automated phenotypic screens using light microscopy and FIB-SEM

Correlative light and electron microscopy (CLEM) combines two imaging modalities, balancing out the limits of one technique with the other. In recent years, Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) has emerged as a flexible method that enables semi-automated volume acquisition at the ultrastructural level. We present a toolset for adherent cultured cells that enables tracking and finding cell regions previously identified in light microscopy, in the FIB-SEM along with automatic acquisition of high-resolution volume datasets. We detect a grid pattern in both modalities (LM and EM), which identifies common reference points. The novel combination of these techniques enables complete automation of the workflow. This includes setting the coincidence point of both ion and electron beams, automated evaluation of the image quality and constantly tracking the sample position with the microscope’s field of view reducing or even eliminating operator supervision. We show the ability to target the regions of interest in EM within 5µm accuracy, while iterating between different targets including unattended data acquisition. Our results demonstrate that executing high throughput volume acquisition in electron microscopy is possible.

Evaluating the Reliability of CBCT Airway Measurement: A Pilot Study

Background Many studies have analyzed treatment effects on the airway using CBCT scans of the airway as evidence, but no study to date has been published that validates a repeatable protocol for acquiring CBCT airway measurements. Our objective is to evaluate a protocol for standardization of CBCT volume acquisition of the airway. Methods Ten participants (6 females and 4 males, median age 30.03 ± 3.53 years) were radiographed 2 times each (T1 and T2, the average time between T1-T2 was 15.8 ± 3.65 days), for a total of 20 CBCT scans. The participants’ head position was placed so that the Frankfort horizontal plane was parallel to the floor. Participants were given specific instructions regarding jaw position, tongue position, swallowing and breathing before each of the 2 scans. Minimum cross-sectional area (MCA) and total volume (TV) of each airway scan were measured between the distal most point of the hard palate (superior limit) and the superior most point of the epiglottis (inferior limit). T1 and T2 were compared to determine the repeatability of our proposed protocol for CBCT airway acquisition. Results There was no statistically significant difference between T1 and T2 measurements for TV and MCA. The average difference between T1 and T2 MCA and TV measurements for each participant was 34.18 ± 30.55 mm2 and 2.51 ± 2.05 cc, respectively. Conclusions Using our proposed standardization protocol, the measurements of MCA and TV of the airway were reliable to within 34.18 ± 30.55 mm2 and 2.51 ± 2.05 cc, respectively.

Non-invasive assessment of central venous pressure in heart failure: a systematic prospective comparison of echocardiography and Swan-Ganz catheter

Assessing hemodynamics, especially central venous pressure (CVP), is essential in heart failure (HF). Right heart catheterization (RHC) is the gold-standard, but non-invasive methods are also needed. However, the role of 2-dimensional echocardiography (2DE) remains uncertain, and 3-dimensional echocardiography (3DE) is not always available. This study investigated standardized and breathing-corrected assessment of inferior vena cava (IVC) volume using echocardiography (2DE and 3DE) versus CVP determined invasively using RHC. Sixty consecutive HF patients were included (82% male, age 54 ± 11 years, New York Heart Association class 2.23 ± 0.8, ejection fraction 46 ± 18.4%, brain natriuretic peptide 696.93 ± 773.53 pg/mL). All patients underwent Swan-Ganz RHC followed by 2DE and 3DE, and IVC volume assessment. On 2DE, mean IVC size was 18.3 ± 5.5 mm and 13.8 ± 6 mm in the largest deflection and shortest distention, respectively. Mean CVP from RHC was 9.3 ± 5.3 mmHg. Neither 2DE nor 3DE showed acceptable correlation with invasively measured CVP; IVC volume acquisition showed optimal correlation with RHC CVP (0.64; 95% confidence interval 0.46–0.77), with better correlation when mitral valve early diastole E wave and right ventricular end-diastolic diameter were added. Using a CVP cut-point of 10 mmHg, receiver operating characteristic curve showed true positivity (specificity) of 0.90 and sensitivity of 62% for predicting CVP. A validation study confirmed these findings and verified the high predictive value of IVC volume assessment. Neither 2DE nor 3DE alone can reliably mirror CVP, but IVC volume acquisition using echocardiography allows non-invasive and adequate approximation of CVP. Correlation with invasively measured pressure was strongest when CVP is > 10 mmHg.

XperCT Sharpening Reconstruction for Cone-Beam Computed Tomography Guided Lung and Bone Interventions

C-arm cone-beam computed tomography (CBCT) is a valuable tool for three-dimensional navigation and mapping in the interventional radiology suite owing to its flexible gantry positioning, real-time three-dimensional volume acquisition, and reduced contrast and radiation use. Reports of CBCT-guided bone and lung interventions are relatively infrequent, however, possibly due in part to the lack of dedicated bone and lung reconstruction algorithms and concerns regarding insufficient lesion conspicuity. Two cases of an ad hoc intraprocedural CBCT sharpening reconstruction are presented in this article.

P6467Pressure-mediated changes in the ventricular septum shape during exercise in patients with pulmonary hypertension: a 3-d echocardiography study

Background The role of interventricular (IV) septum in mediating the mechanical interaction between the two ventricles is well recognized. This interaction is promoted by several structural and hemodynamic determinants. We aimed at assessing the geometrical RV changes in patients with pulmonary hypertension (PH) compared to normal, evaluating RV curvature at rest and during stress testing addressing the specific role of pressure-induced changes in IV septum curvature. Methods Thirty-four subjects (15 controls; 19 PH patients) underwent RV real-time 3D full volume acquisition at rest and during exercise-echo. The 3D data were analysed off-line using the 4D RV TomTec software. The 3D mesh of the RV model was post-processed using a custom developed software. The value of mean regional curvature was assessed for the interventricular septum (IVS) at end-diastole (ED) and at end systole (ES). Results In controls the IVS curvature, assessed at end-diastole (ED) and end-systole (ES), was significantly (p<0.001) more concave (at rest −0.31±0.06 at ED, and −0.29±0.06 at ES; during exercise −0.28±0.09 at ED, and −0.28±0.08 at ES) than in PH patients (at rest −0.09±0.14 at ED, and −0.09±0.11 at ES ; during exercise −0.05±0.18 at ED, and −0.02±0.18 at ES). There was no significant variation in any of RV IVS curvatures between rest and exercise or between ES and ED. In PH subjects, IVS curvature at ES weakly increased from −0.09±0.11 (SD) at rest to −0.02±0.18 during exercise (p=0.179).Nonetheless, the degree of IVS curvature was strongly related to systolic pulmonary artery pressure (PASP), both at rest (r=0.743 at ES, p<0.01; r=0.794, p<0.001 at ED) and during exercise (r=0.823 at ES, p<0.0001; r=0.812 at ED, p<0.0001). Conclusions These data provide new perspectives on how the interventricular septum morphology adapts during exercise in PH patients vs controls. Changes in IVS curvature are linearly related to pulmonary pressure changes and occur with different slope (rest-exercise) in PH vs controls.