Recently, several studies have proposed methods for measuring cuffless blood pressure (BP) using finger photoplethysmogram (PPG) signals. This study presents a new BP estimation system that measures PPG signals under progressive finger pressure, making the system relatively robust to errors caused by finger position when using the cuffless oscillometric method. To reduce errors caused by finger position, we developed a sensor that can simultaneously measure multi-channel PPG and force signals in a wide field of view (FOV). We propose a deep-learning-based algorithm that can learn to focus on the optimal PPG channel from multi channel PPG using an attention mechanism. The errors (ME ± STD) of the proposed multi channel system were 0.43±9.35 mmHg and 0.21 ± 7.72 mmHg for SBP and DBP, respectively. Through extensive experiments, we found a significant performance difference depending on the location of the PPG measurement in the BP estimation system using finger pressure. This method is suitable for the long-term observation of BP changes in patients admitted to the intensive care unit (ICU). However, there is a risk of infection owing to its invasive nature2. Non-invasive BP measurement methods include cuff-based3 and cuffless methods. Cuff-based BP measurement methods use an electronic sphygmomanometer device and are widely accepted as the gold standard because they can achieve relatively high accuracy. Moreover, users can easily measure BP at home without the help of medical staff4. However, there are disadvantages in that the user feels uncomfortable because of the applied pressure. Furthermore, an electronic sphygmomanometer device is not easily portable.The most accurate blood pressure (BP) measurement method involves a medical catheter6, in which the BP is predicted using various biomedical signals, such as photoplethysmogram (PPG) and electrocardiogram (ECG). The pulse transit time (PTT) or pulse arrival time (PAT) can be calculated using simultaneously measured PPG and ECG signals5. They are calculated using the time difference between the peaks of the two signals measured by the sensor at two different points in the artery. Although several studies have used the correlation between PTT or PAT and BP7 for BP prediction, they are not suitable for mobile devices such as smartphones or smart watches because they require two sensors in different locations to measure both PPG and ECG signals. One method for overcoming this disadvantage is to predict the BP using pulse wave analysis (PWA) from PPG signals. A PPG signal is a periodic pulse-wave signal that is correlated with the cardiovascular system. Therefore, some researchers have extracted engineered features such as height and width within the pulse wave and used them to predict BP8. However, extracting accurate engineered characteristics is challenging because the characteristics of the cardiovascular system differ from person to person owing to factors, such as age, disease, and drugs administered9.Recently, several studies have proposed cuffless BP measurement methodset al. proposed the measurement of BP on a smartphone using the finger pressure method10, which estimates BP by using the change in the PPG envelope during blood vessel constriction caused by a gradual increase in finger pressure on the PPG sensor. However, because the sensor uses a single-channel PPG, the accuracy of BP prediction can be significantly affected by the position of the finger on PPG sensor.Recently, Mukkamala et al.10. The proposed PPG sensor simultaneously measures multi-channel PPG signals and a finger pressure signal, while the subject’s fingertip gradually pressurizes the sensor for 40 s. The measured multi-channel PPG and finger pressure signals represent progressive pressure characteristics and have characteristics similar to those of oscillometric wave (OMW) signals and cuff pressure signals used in cuff-based BP measurement methods. Moreover, the multi-channel extension in PPG signal measurement is expected to alleviate the variation in the position of the finger on the PPG sensor. Second, we proposed a deep-learning-based BP estimation system using multi-channel PPG and finger pressure signals. The proposed deep learning-based BP prediction system consists of two parts. First, a convolutional neural network (CNN)-based model was designed, which extracts channel-specific features for BP estimation from the multi channel PPG and finger pressure signals. Second, a multi-channel attention network for improved BP estimation accuracy was proposed, which combined the latent features obtained from the single-channel BP estimators to produce a new attention-weighted feature. The combined feature was subsequently used for the final BP estimation.In this study, we propose a novel approach to cuffless BP estimation. The contributions of this study comprise two main aspects. First, we developed a sensor that can acquire multi-channel PPG signals with different wavelengths using a finger pressure method similar to that used by Mukkamala The oscillometric method for measuring BP uses the changes in PPG amplitudes caused by the occlusion of blood vessels. To accurately measure PPG signals at high signal-to-noise ratios, the optical path of the sensor must include blood vessels. However, because fingers have a complex vascular structure, there are small arteries on both sides of the finger and across the tip of the nail. Therefore, it is difficult to accurately form an optical path that includes blood vessels using a single-channel PPG sensor. To overcome this challenge, we developed a multi-channel PPG sensor with a wide field-of-view (FOV).The proposed PPG sensor comprises the following components, as shown in Fig. entclass1pt{minima11 which was describing relevant guidelines for clinical investigation of Non-invasive Sphygmomanometers including subject requirements , reference readings . All examinees provided informed consent before the measurements were conducted.Clinical trials were conducted separately at two different sites: the MONIKI Hospital in Russia and Samsung Medical Center in Korea. Dataset1 was collected at the MONIKI Hospital and contained 1,450 cases from 290 participants; it was used to train the proposed BP estimation system. Dataset2 was collected at the Samsung Medical Center and contained 865 cases from 186 participants; it was used for training and testing. Each case included 40s synchronized 9-channel PPG and force signals. And reference BP obtained from two medical staff using auscultation was also included. Data collection was approved by the ethical committee of Samsung Medical Center (IRB Protocol No: 2020-06-065). The study design followed the International Standard (ISO 81060-2)For clinical trials, the proposed multi-channel PPG system was used in compliance with standard protocols ISO 81060-2). The reference BP was measured by two medical staff members using the auscultation method, and five measurements were conducted for each subject. The participants took a break of at least 5 min between measurements to ensure stability. The finger was then placed on a pre-marked guideline. After the measurement started, they were asked to gradually press the sensor with their index finger for 40 s while watching the pressure increase guide displayed on the computer screen. Table 060-2. ThWe used dataset1 of 290 participants and dataset2 of 186 participants for the training, validation, and testing of the BP estimation system. We divided the training, validation, and test data such that there were no overlapping participants. Dataset1 was used only for training and validation, whereas dataset2 was used only for training and testing. we split dataset1 with 183 participants for training and 107 participants for model validation. In addition, dataset2 was divided into file-folds without overlapping participants, one-fold was used for testing and the remaining folds were used for training, and each of the five-folds was tested in turn. Therefore, we performed model verification through a five-fold cross-validation of dataset2. All the five-fold BP estimation results obtained using dataset2 are presented in.12 was used with To train the nine single-channel CNN-based feature extractors and multi channel attention mechanism, the Adam optimizerWe used the mean of the error (ME), standard deviation of the error (STD), and Pearson’s correlation coefficient (r) as the evaluation metrics for BP prediction. In addition to evaluating the overall BP estimation system, the BP estimation performance of the CNN-based feature extraction model for a single PPG and attention mechanism were compared and analyzed.13 or the University of Queensland Vital Signs dataset14. These public datasets contain unpressurized single-channel PPG and ECG signals. Although studies16 have used self-made datasets, these usually contain unpressurized single-channel PPG signals or ECG signals. In this study, we designed a BP prediction system using the signals obtained from the proposed multi channel PPG and finger pressure sensors. Because our self-made dataset contains multi-channel PPG signals applied under pressure and finger pressure signals, a direct comparison with other BP estimation studies is difficult. Therefore, we analyzed the proposed multi-channel PPG-based BP estimation system and its components.Most studies that have developed a BP prediction model use Physionet’s multi-parameter intelligent monitoring in intensive care (MIMIC) online waveform databaseTables Table The attention mechanism of our proposed BP estimation system is important for improving the BP estimation performance. Figure We verified that the proposed multi-channel attention mechanism could improve the BP prediction performance of single-channel models. The attention mechanism could predict BP more accurately by considering the importance of latent features extracted from multiple single-channel PPG signals. In this subsection, we confirm the effectiveness of the attention mechanism by changing its attention method. Table We compared the BP estimation accuracy according to the input signal combination in the single-channel BP estimation model for SBP and DBP, respectively. We compared the performance when only PPG, first and second differential signals, and finger pressure signals were used, the performance when envelope signals and finger pressure signals were used, and the performance when both were used. As shown in Table 17 for SBP and DBP estimation are shown in Figs. To further verify the proposed BP estimation system, the scatter plot and Bland–Altman plot19. Because the size of the training dataset is known to substantially affect the performance of neural-network-based BP prediction models20, additional performance improvements can be expected by collecting additional training datasets using the developed sensor. However, clinical data collection is hampered by high cost, excessive time, and other limitations. Including inter- and intra-individual BP variation is important for the evaluation of cuffless devices, but difficult to obtain21. Our acquired dataset only acquired BP under static conditions and did not consider BP variation within each individual. Additionally, demographics  are often used as additional inputs to BP prediction models21. However, our proposed model does not take advantage of this to relieve the hassle of requiring users to enter demographic information.In this study, we developed a multi-channel PPG sensor that senses multi-channel PPG signals and finger pressure signals and proposed a cuffless BP estimation system. The acquired multi-channel PPG signals were obtained by placing a finger on the proposed sensor and gradually applying pressure. Using the developed sensor, dataset1 and dataset2 were collected from the MONIKI Hospital in Russia and the Samsung Medical Center in Korea, respectively. Dataset1 and dataset2, which contain 290 and 186 participants, respectively, are small datasets compared to the MIMIC online waveform database and University of Queensland Vital Signs datasets, which many researchers use to train their BP predictive models22. The proposed BP estimation system was verified using five-cross-validation by setting dataset2 as the target dataset. we used regularization terms and dropout techniques to prevent model overfitting. and we showed the performance of the model through 5-fold cross validation. at this time, all performances for each 5-fold were shown. we applied the methods to prevent model overfitting with insufficient clinical data, as in other studies. If we apply a domain adaptation technique23 that can achieve better performance for the target dataset while reducing the interval between datasets in different domains24, we believe that we can obtain a more accurate estimated BP from dataset2.In our study, dataset1 and dataset2 had slightly different conditions, such as the data acquisition environment, location, and some sensor specifications. As mentioned earlier, when the BP estimation system is trained using two datasets with different domains, it is difficult to expect a high accuracy in the target dataset25, the BP estimation error should be within 5±8mmHg. The proposed BP estimation system satisfied the AAMI criterion for DBP, and the SBP was also close to the AAMI criterion. The accuracy of our proposed BP estimation system can be improved if more data are collected, and the domain mismatch in the datasets collected in different environments between datasets owing to features of different domains are resolved. We plan to improve the deep-learning-based BP estimation system to obtain more accurate predicted BP values by applying domain adaptation techniques to our two datasets with different characteristics.According to the Association for the Advancement of Medical Instrumentation (AAMI) standardWe propose a novel BP prediction system with an attention mechanism that uses multi-channel PPG signals and a finger pressure signal. The proposed BP prediction system can learn to extract features from raw PPG and finger pressure signals using an end-to-end deep-learning method without relying on human-engineered, hand-crafted feature extraction methods. Moreover, an attention mechanism allows the proposed system to effectively combine features extracted from each PPG channel.27. Therefore, we modeled the BP prediction system by adding the envelope signal and the first- and second-order differential signals. A block diagram of the signal preprocessing method is shown in detail in Fig. Because the acquired raw PPG and finger pressure signals contained noise components, filtering was applied to remove noise. Specifically, to use various input signals for the feature extraction model, preprocessing steps were performed to obtain the filtered PPG signal envelope and differential signals . Previous studies on BP prediction using PPG signals showed that using the first- and second-order differential signals of PPG in addition to the PPG signal, the BP prediction model can more accurately predict BP by modeling various informationY) to train the proposed BP prediction system, where Through the signal preprocessing described above, we constructed a dataset 28. Thus, we constructed three parallel input streams of the CNN model such that the features were extracted for each of the X and Z, denote the input and extracted features, respectively, and iZ, is then introduced to a fully connected layer with sigmoid nonlinearity, producing the final latent feature containing various types of information extracted from different input signals. Residual connections resolve the vanishing gradient problem29 when training the feature extraction model. After the CNN-based feature extraction models are trained for each PPG channel, the attention mechanism can be trained to combine multi channel features for BP prediction.The CNN successfully learns the relationship between neighboring data points through a convolution operation and can compress information from the input signal through a pooling layerN denotes the number of samples; Z is the final latent feature, and iTo train the feature extraction model, the last output layer produces the estimated BP 31 and natural language processing32. Attention mechanisms are neural networks that focus on important regions. We extracted each feature from a CNN-based feature extraction model using multi channel PPG and finger pressure signals. However, because the position of the finger placed on the proposed multi channel PPG sensor and the characteristics of the fingers may be different for each user, the importance of each channel for BP estimation may also differ for each user. Therefore, we applied an attention mechanism to the proposed BP estimation system for adaptively weighing channel-wise features according to their importance in estimating the BP for each user.Recently, attention mechanisms have proven effective in many fields, such as speech recognitionb are the trainable weights and biases, respectively. Subsequently, from the obtained score, As shown in Fig. In this study, we developed a multi-channel PPG sensor that acquires multi-channel PPG signals at different wavelengths. Moreover, we devised a deep-learning-based BP estimation system that can predict BP from multi-channel PPG signals acquired from the proposed sensor and finger pressure signal. The proposed BP estimation system can extract features without human engineering and accurately predict the BP through an attention mechanism. Through attention weight analysis, we confirmed that the attention mechanism can improve the prediction performance of hypertension and hypotension. Because the proposed deep-learning-based BP estimation system is a cuff-free and calibration-free method, it is possible to monitor BP regularly and has the potential to diagnose hypertension at an early stage. The proposed BP estimation system can potentially enable regular BP monitoring of multiple users through mobile devices, such as smartphones or smart wristwatches.


Sensory cortical areas are robustly modulated by higher-order cortices. Our previous study shows that the anterior cingulate cortex (ACC) can immediately and transiently enhance responses in the mouse auditory cortex (ACx). Here, we further examined whether strong activation of ACC neurons can induce long-term effects in mice of both sexes. To our surprise, only stimulation of cell bodies in the ACC, but not ACC-to-ACx terminal activation, induced long-term enhancement of auditory responses in the ACx. Anatomical examination showed that the ACC indirectly projects to the ACx via the rhinal cortex (RCx). High-frequency stimulation of ACC-projecting terminals to the RCx or RCx-projecting terminals to the ACx induced a similar effect as the cell body activation of ACC neurons, whereas silencing the RCx blocked this long-term enhancement. High-frequency stimulation of ACC projections to the RCx also induced long-term augmentation of sound-evoked flight behavior in male mice. These results show that the ACC promotes the long-term enhancement of auditory responses in the ACx through an indirect pathway via the RCx.SIGNIFICANCE STATEMENT In this study, we demonstrate that the anterior part of the anterior cingulate cortex (ACC) evokes long-term enhancement of auditory responses in the auditory cortex (ACx) when it is strongly activated. Importantly, instead of a direct projection, we show that the ACC implements this effect via an indirect pathway through the lateral rhinal cortex using a series of physiological, optogenetic, anatomic, and behavioral experiments. Along with a short-term effect, this long-term enhancement induced by an indirect ACC-to-ACx projection could increase the odds of survival when animals are faced with threats after a significant event. Primary sensory cortical areas  are adaptive processors that are subject to robust top-down modulation by high-order brain areas. These areas are also strongly influenced by various statuses of the organisms, such as arousal, attention, and perceptual learning. Regions of the prefrontal lobe that send top-down projections to the sensory cortex are known to play a critical role in high-level cognitive functions, such as attentional control and working memory . In partTherefore, we first examined whether long-lasting potentiation of auditory responses  in the ACx could be induced by high-frequency activation of ACC cell bodies or ACC-projecting terminals in the ACx. To our surprise, only the activation of ACC cell bodies caused long-term enhancement of auditory responses. Hence, we hypothesized that the ACC induces this effect through other brain areas. As previous studies show that the temporal cortex, specifically the rhinal cortex (RCx), possesses the ability to facilitate the formation of LTP and associative memory in the neocortex , we furtad libitum access to food and water. Behavioral experiments were conducted during the dark cycle.All experimental procedures were approved by the Animal Subjects Ethics Sub-Committee of the City University of Hong Kong. Electrophysiological and anatomic experiments were conducted using adult male and female C57BL/6J mice. Behavioral experiments were conducted using only adult male C57BL/6J mice, considering that female mouse behavior could be influenced by the estrous cycle. Animals were recruited at ages from 8 to 12 weeks. Animals were housed under a 12 h dark/12 h light cycle  at a stable temperature (23°C-25°C) with Adeno-associated viruses (AAVs) were purchased from Addgene , BrainVTA (AAV9-hSyn-tdTomato-WPRE-hGHpA [#PT-1206-AAV9]) and Taitool Bioscience .For mice in electrophysiological experiments, AAV9-Syn-ChrimsonR-tdTomato (4.7E + 12 vg/ml) or AAV9-hSyn-tdTomato-WPRE-hGHpA (5.12E + 12 vg/ml) was injected into two locations of the aACC in the left hemisphere , and AAV9-CaMKIIα-Chronos-GFP (5.6E + 12 vg/ml) or AAV9-CaMKIIα-eGFP (4.7E + 12 vg/ml) was injected into two locations of the lateral RCx (lRCx) in the left hemisphere .Before surgery, animals were administered intraperitoneal pentobarbital sodium . An animal was fixed in a stereotaxic device (RWD), and the scalp was incised after cleaning twice with 75% alcohol. The skull was washed with saline and then adjusted in position to level Bregma and Lambda sites as well as left and right sides. Craniotomies with diameters of ∼0.5-1 mm were created above the injection sites. AAV was loaded in a pipette with a fine tip mounted on the Nanoliter 2000/Micro4 system (World Precision Instruments). The injection pipette was slowly advanced to the target depth and paused for 5 min before the infusion. The infusion speed was 25 nl/min for aACC injection and 50 nl/min for lRCx injection. The pipette was slowly withdrawn 10 min after the infusion was complete. Four to 5 weeks were allowed for the expression of the virus.White noise  stimuli were digitally generated by Synapse controlled RZ6 processor  and delivered through a free-field magnetic speaker . The sound pressure level was calibrated with a sound level meter  with the A-weighing curve. The meter faced the speaker at a distance of 10 cm during measurement. During the experiment, the speaker was fixed at the same place facing the animal's right ear and 10 cm away. To choose a proper sound intensity that induced mild auditory responses, we first roughly determined the auditory response threshold in the mouse ACx by presenting noise stimuli at various sound intensities . The threshold was defined as the intensity at which ACx neurons started to show obvious auditory responses on examination of their raster plots. We selected an intensity 10 dB higher than the threshold to ensure that reliable auditory responses were evoked with sufficient room for modulation by manipulations. Laser stimulation was generated by a laser source (Inper #B1465635 Hangzhou) triggered by the Synapse-controlled RZ5D processor (Tucker-Davis Technologies). A fiber optic cannula  connected to an optical fiber patch cable was used to deliver the laser to the target area. A train of 40 laser pulses with 5 ms duration at 40 Hz was adopted as the high-frequency laser stimulation (HFLaser) protocol, whereas a train of 40 laser pulses with 5 ms duration at 1 Hz was adopted as the low-frequency laser stimulation (LFLaser) protocol. The laser power was 5 mW for neuronal cell body activation and 20 mW for neuronal projection activation.For extracellular recordings in the ACx and lRCx, mice were first injected with urethane sodium , and anesthesia was maintained with periodic supplements throughout the surgery and neuronal recordings. A heating pad was used to keep the core body temperature of animals at 37°C. Mice were mounted in a stereotaxic device, and a midline incision was made in the scalp. The muscles above the ACx and lRCx were removed, after which a craniotomy was performed above the ACx. For the lRCx, the craniotomy was performed at the juncture of the temporal, occipital, and interparietal bones to expose the caudal rhinal vein and transverse sinus . A 32-chm. Glass capillaries  were pulled using a micropipette puller  with proper parameters. The impedance of the glass pipette electrode filled with 1 m NaCl was tested with a Multiclamp 700B microelectrode amplifier (Molecular Devices). A fiber optic cannula was held just on the surface of the aACC to present 40 pulses of laser stimulation at different frequencies  every 10 s.For aACC neuronal responses to laser stimulation, to avoid any photoelectric artifacts, MUAs evoked by laser stimulation were recorded by glass pipette electrodes with an impedance of 1 For MUAs, a 5 ms time bin was adopted to calculate peristimulus time histograms (PSTHs). The response window was 0-0.1 s after the noise onset. Responses were averaged every 10 trials and then normalized as the percentage of change referencing each channel's mean response before laser stimulation. For fEPSPs, we recorded only one site (the deepest site) from each shank as a representative site considering the similar fEPSP origin for one cortical column. fEPSPs were averaged every 30 trials. The slope was measured by fitting a line to the initial linear phase of the fEPSP response. Slopes were normalized as the percentage of change referencing each site's mean slope before laser stimulation. All MUA and fEPSP signals were extracted and processed with MATLAB (The MathWorks) and then analyzed and visualized with R software (R Foundation).m, Sigma-Aldrich #M1523) into the left lateral RCx or lidocaine  into the left aACC. To label the diffusion range, we injected fluorescent muscimol (Fisher Scientific #M23400) into the lRCx and fluorescein  (A 10 µl syringe (Hamilton) coupled with a plastic tube and corresponding injector (RWD) was controlled using a syringe pump (KD Scientific) to infuse muscimol   into theFor retrograde tracing, cholera toxin subunit B (CTB) with AlexaFluor-488 conjugate  was injected into the left ACx . CTB with AlexaFluor-647 conjugate  was injected in the left lRCx .For collateral projection labeling, AAVretro-hSyn-Cre-WPRE-hGH (1.83 + 13 vg/ml) was injected into the ACx . After 1 week, AAV8-Syn-DIO-mCherry (5.3 + 12 vg/ml) was injected into the aACC .For anterograde tracing, the left aACC (the same coordinates mentioned above) was injected with AAV1-hSyn-Flpo-WPRE-pA . After 1 week, the left lRCx (the same coordinates mentioned above) was injected with AAV9-hEF1a-fDIO-eYFP-WPRE-pA .https://imagej.net/Fiji) (https://atlas.brain-map.org/). The Cell Counter plugin in Fiji was used to count cell numbers. To measure the projection intensity of ACC neural fibers, the FeatureJ plugin was used in Fiji. Fiber-like signals were extracted using a Hessian filter, and the fewest eigenvalues were selected when converting the raw images to Eigen images. The intensity of neural projections was assessed as the pixel density after converting Eigen images to binary images in Fiji  and sequentially perfused with 30 ml of 1× PBS followed by 30 ml of 4% (w/v) PFA. The brains were subsequently removed and postfixed in 4% PFA at 4°C overnight. After cryoprotection of the brains with 30% (w/v) sucrose, coronal sections (70 μm) were cut on a cryostat (Fisher Scientific #HM525NX). The brain slices were mounted on a glass slide with 70% glycerol in PBS, and fluorescent images were taken by a Nikon Eclipse Ni-E upright fluorescence microscope  and confocal microscope . For imaging signal analysis, including quantification of neuron number and projection intensity, we used Fiji software (et/Fiji) . To calc in Fiji .AAV9-Syn-ChrimsonR-tdTomato or AAV9-hSyn-tdTomato-WPRE-hGHpA was injected into the left aACC, and 4-5 weeks were allowed for virus expression. On the optical fiber implantation day, dexamethasone  and carprofen  were administered at least 1 h before anesthetization induced by pentobarbital sodium (80 mg/kg). A mouse was then fixed in a stereotaxic device (RWD), and the scalp was incised. After cleaning and leveling the skull, small holes were drilled bilaterally to reach the lRCx. The dura was removed, and optical fibers  were slowly inserted. Kwik-cast gel (World Precision Instruments) was applied around the cannula to seal the drilled skull. A thin layer of adhesive cement  was then applied to the skull. Fibers were fixed to the skull with dental cement . Finally, a long screw was attached to the skull  with dental cement for later head-fixed experiments.We used a sound-evoked flight behavioral paradigm . Animalst tests, two-way repeated ANOVA, and two-way mixed ANOVA, were conducted in SPSS 26 (IBM). Statistical significance was defined as p < 0.05 by default  and the ACx (A). To confirm the effectiveness of the virus and responsiveness of aACC neurons, a glass pipette electrode along with an optical fiber was inserted into the virus injection region of anesthetized mice (B). Forty laser pulses at different frequencies  were applied. aACC neuron responses faithfully followed up to 40 Hz . Therefore, the HFLaser protocol (forty 647 nm laser pulses at 40 Hz) was used to strongly activate the aACC, and the LFLaser protocol (forty 647 nm laser pulses at 1 Hz) was used as a control. An electrode array was inserted into the ACx to record fEPSPs and MUAs, and an optical fiber was placed at the aACC to shine the laser stimuli (A). A series of white noises  was presented. To identify the auditory response threshold in the ACx, neuronal responses to 100 ms noise stimuli at different sound intensities  were examined. We selected an intensity 10 dB higher than the threshold to ensure that reliable auditory responses were evoked, and there was sufficient room for modulation following manipulations. After a baseline recording of 20 min, the HFLaser or LFLaser protocol was applied, and auditory responses were monitored for another hour. The LFLaser protocol did not induce a significant change in auditory responses in terms of fEPSPs or MUAs . By contrast, after the HFLaser protocol, both the slopes of fEPSPs and firing rates of MUAs gradually increased , indicating that only high-frequency activation of aACC neuronal cell bodies promoted long-term enhancement of auditory responses in the ACx. The slopes of fEPSPs started to increase earlier than the firing rates of MUAs, possibly because of the nonlinear relationship between the input and output of neurons in the ACx .Our recent study shows that a single pulse activation of aACC terminals in the ACx immediately potentiates auditory responses in the ACx . Here, t the ACx A. To conzed mice B. Forty to 40 Hz C,D. Ther stimuli A. A seri the ACx . In a coG). To our surprise, the HFLaser protocol did not induce significant changes in either fEPSPs or MUAs in response to noises .To rule out the effect of other areas triggered by the stimulation of aACC neuronal cell bodies, in the next experiment, the optical fiber was shifted to the ACx to activate aACC-to-ACx-projecting terminals G. To ourTherefore, once strongly activated, the aACC possessed the ability to potentiate auditory responses in the ACx in the long-term. As this effect was not mediated by direct projections from the aACC to the ACx, other areas were potentially involved.A). High levels of tdTomato signal were observed in both the ACx and lRCx regions, implying that the aACC induces long-term enhancement of auditory responses in the ACx by activating the lRCx. Retrograde labeling by CTB conjugated with a fluorescent tag Alexa-647 injected into the RCx further confirmed the existence of aACC-to-RCx projections . As we previously focused on the entorhinal part of the RCx, we next investigated whether the lRCx possesses a similar ability. A virus (AAV9-CaMKIIα-Chronos-GFP) expressing a blue light-sensitive cation channel (Chronos) that can be activated by a 473 nm laser was injected into the lRCx of WT mice to infect excitatory neurons through the CaMKIIα promoter . Strong expression of GFP was found in the ACx 4 weeks later . An electrode array together with an optical fiber was inserted in the ACx to record fEPSPs and MUAs, a train of forty 473 nm laser pulses at 40 Hz was applied in the ACx to activate the lRCx to ACx-projecting terminals. After the HFLaser protocol, similar to ACC neuronal cell body activation, a gradual increase in auditory responses in the ACx was observed . Again, an increase in fEPSP slopes led to an increase in the firing rates of MUAs, implying the same underlying mechanism as ACC neuronal cell body activation. In a control experiment in which the virus without the Chronos light-activated cation channel (AAV9-CaMKIIα-eGFP) was injected, no significant change was observed after the HFLaser protocol . These results show that the lRCx is a potential site that endows the aACC with the ability to promote the long-term enhancement of auditory responses in the ACx.In our previous studies, we demonstrated that the RCx  promotes the formation of LTP and auditory associative memory in the ACx through its cholecystokinin-positive projections . Thus, wFig. 3A). Most labeled neurons in the aACC projected only to the ACx  or to the lRCx . ACx-projecting neurons concentrated in deep layers, whereas lRCx-projecting neurons were located in both superficial and deep layers . To further validate the separation of these two groups of neurons in the aACC, AAVretro-hSyn-Cre was injected into the ACx of WT mice followed by injection of a Cre-dependent virus (AAV8-hSyn-DIO-mCherry) into the aACC to label neurons projecting to the ACx . Similar to CTB488 labeling, neurons in deep layers of the ACC expressed mCherry . For the projections, high levels of mCherry signals were detected in the ACx and amygdala . By contrast, signals were sparse in the RCx . Therefore, two groups of neurons in the aACC appeared to separately govern the ACx and lRCx. The following question would be whether those RCx neurons receiving projections from the aACC send axons to the ACx. To answer this question, an anterograde trans-synaptic virus (AAV1-hSyn-Flpo-WPRE-pA) was injected into the aACC region of WT mice followed by injection of an Flp-dependent virus (AAV9-hEF1a-fDIO-eYFP-WPRE-pA) into the lRCx 1 week later to label lRCx neurons receiving projections from the ACC . Four weeks later, eYFP fluorescent signals were found in the ACx, representing direct projections from lRCx neurons innervated by the aACC. These findings demonstrate the existence of an indirect pathway from the aACC to the ACx via the lRCx in addition to a direct projection from the aACC to the ACx.To understand how the lRCx mediates this potentiating effect, we investigated the anatomic connections among the aACC, lRCx, and ACx. Distributions of retrogradely labeled neurons were examined after CTB488 and CTB647 were infused into the ACx and lRCx, respectively, in WT animals A. Most lA). Muscimol infusion silenced neural activity in the target region for >1 h (B), which allowed us to test the effectiveness of the HFLaser protocol in the aACC while inhibiting neuronal activity in the lRCx. Immediately after muscimol injection , neuronal responses of noise-evoked fEPSPs and MUAs in the ACx were recorded (A). Following a 20 min baseline recording, the HFLaser protocol was applied in the aACC, and responses in the ACx were monitored for another hour. After the lRCx was silenced, high-frequency activation of aACC neuronal cell bodies no longer enhanced auditory responses in the ACx for both auditory-evoked fEPSPs and MUAs . This loss-of-function experiment demonstrates that an intact lRCx was indispensable for the ability of the aACC to facilitate auditory responses in the ACx long-term.After showing the existence of this indirect pathway from the aACC to ACx via the lRCx anatomically, the next step was to examine whether the lRCx participates in the induction of long-term enhancement in the ACx by the HFLaser protocol in the aACC physiologically. Muscimol, a GABA A receptor agonist, was infused into the lRCx through a glass pipette A. Muscimfor >1 h B, which recorded A. FollowA). The HFLaser protocol evoked significant neuronal activity in the lRCx (B), showing the excitatory nature of the projection from the aACC to the lRCx. In the next experiment, the electrode array was inserted into the ACx to monitor auditory responses, and the optical fiber was moved to the lRCx to activate aACC-projecting terminals in the lRCx by the HFLaser protocol while cell bodies in the aACC were silenced by lidocaine (C). The same volume of fluorescein was administered to check the diffusion range of lidocaine, which overlapped well with the virus expression region in the aACC. The HFLaser protocol in the lRCx induced a similar long-term enhancement of auditory responses as aACC neuronal cell body excitation , which supports the hypothesis that the aACC implements this enhancing effect through the lRCx. In mice in which a control virus (AAV9-Syn-tdTomato) was injected, no significant change was observed after the HFLaser protocol .To further confirm the participation of the lRCx, we injected AAV9-Syn-ChrimsonR-tdTomato into the aACC region and inserted the electrode array into the lRCx while placing an optical fiber at the virus injection site aACC, A. The HFthe lRCx B, showinidocaine C. The saA). A sound-evoked flight paradigm was adopted to measure the flight response to noise (B). First, 5 s noises at 30, 50, 70, and 90 dB were presented, and animals' running speeds were measured as baseline responses. Next, four pairings of the HFLaser protocol and noise were applied to induce the presumed long-term enhancement of auditory responses in the ACx (B). One hour later, the same series of 5 s noises was presented to examine the long-term effect. As an example, for 90 dB, the HFLaser protocol applied in the lRCx significantly facilitated sound-evoked flight responses . In mice injected with the control virus (AAV9-Syn-tdTomato), no effect was observed after the HFLaser protocol in the lRCx . Group data show that the HFLaser protocol in the lRCx significantly elevated the speed-to-intensity psychometric curve in mice injected with AAV9-Syn-ChrimsonR-tdTomato  but not in mice injected with the control virus . Therefore, when aACC-to-lRCx projecting terminals were excited at high frequency, long-term enhancement of auditory responses was also induced behaviorally.Thus far, our study demonstrates that the aACC indirectly induced the long-term enhancement of auditory responses in the ACx via the lRCx anatomically and physiologically. Finally, we conducted a behavioral experiment in which we optogenetically activated aACC-projecting terminals in the lRCx and tested whether this manipulation could enhance sound-evoked flight responses long-term. AAV9-Syn-ChrimsonR-tdTomato was injected into the aACC region, and optical fibers were implanted into both sides of the lRCx A. A sounto noise B. First, the ACx B. One hoG). Along with the short-term effect promoted by ACC direct projections, the survival odds of animals may be elevated, as animals can have speedier responses when facing ensuing threats after a significant event. The detailed operating mode of direct and indirect routes is still unknown. We suppose that the two pathways can be activated at the same time when mice face a crucial incident. However, their engagement under usual circumstances may depend on the level of significance. With low to moderate levels, only the direct pathway is activated, and the high level would also energize the indirect pathways.Previous study showed that the activation of ACC projections by a single pulse of laser in the ACx potentiated auditory responses transiently . Here, wFig. 7C-I). Nevertheless, we cannot rule out the possibility that the long-term change occurred in the lRCx, which drove the change in the ACx, although the brief recording in the lRCx did not show this tendency . The same issue pertains to when we directly stimulated aACC neuronal somas to induce a long-lasting enhancement in the ACx . For instance, the ACC is involved in directing attention toward important sensory stimuli . EnhanceAs the effect generated by this indirect aACC-to-ACx projection can last for a long time, it would intensify the overexcitement of the auditory system triggered by the direct projection from the ACC under pathologic conditions. The combined effect would prolong and exaggerate symptoms related to the overexcited system. Further studies showing how this pathway contributes to the development of related brain disorders would help increase our understanding of pathogenesis and the design of new treatments.


Introduction The current reported mortality rate for elderly neck of femur fractures (eNOFF) is relatively high in the UK. eNOFF patients commonly suffer from associated cardiovascular co-morbidities and tend to have fragile physiological states and poor physiological reserves. Although some studies have shown a potential link between blood transfusion and mortality in eNOFF patients, there is no general consensus on this matter. Therefore, our study aims to explore the possible association between blood transfusion and length of hospital stay (LOHS) as well as short- and long-term mortality rates in eNOFF patients by reviewing the practice of blood transfusion.Methods This retrospective study was conducted at Wrexham Maelor Hospital, which is part of the Betsi Cadwaladr University Health Board (BCUHB), Wales. The study included patients who were 65 years of age or older and presented with neck of femur fractures. Only patients who required surgical intervention were included, and those managed non-operatively were excluded from the study. The statistical analysis was performed using IBM SPSS Statistics for Windows, Version 25.0 . Furthermore, unpaired t-tests and log-rank (Mantel-Cox) tests were performed to compare the groups that received blood transfusions. Results During the study period, a total of 501 eNOFF patients were included in the primary cohort of the study, with a mean age of 81 years (ranging from 65 to 102). The majority of the patients were female (n=340). Of the 501 patients, 79 (15.8%) received a blood transfusion during their treatment. Around 52.9% of the eNOFF patients were categorized as American Society of Anesthesiologists (ASA) III, but there was no statistically significant difference in the requirement of blood transfusion between patients in ASA III, II, and IV categories, as compared to ASA I. Additionally, the mean time to surgery was higher in patients who received a blood transfusion (35.8 hours), and this difference was statistically significant (p=0.035).Moreover, the average LOHS after surgery for eNOFF was longer in patients who needed peri-operative blood transfusion (22 days), and this difference in the means was statistically significant (p=0.022). At the one-year post-surgery mark, mortality was higher in the transfused group (33%), and long-term five-year mortality rates were also higher in this group (63.2%). Conclusion Peri-operative blood transfusion may confer certain benefits in the management of eNOFF ptients. However, it should not be regarded as a panacea for improving long-term outcomes. The decision to administer blood transfusion must be made on a case-by-case basis, with careful assessment of individual clinical indications, and the potential risks and benefits taken into consideration. To achieve optimal clinical outcomes, close monitoring and follow-up of eNOFF patients, both in the short-term and long-term, are essential. Elderly neck of femur fracture (eNOFF) is one of the most common orthopaedic injuries encountered worldwide by orthopaedic surgeons. The annual incidence of eNOFF in the United Kingdom (UK) is 70,000-75,000 . Thus, iThe current reported 30-day mortality for the eNOFF is 8.3% in the UK . eNOFF pThere have been reports of increased morbidity and mortality secondary to increased rates of blood transfusion in eNOFF patients . After aThough some studies have shown an association between blood transfusion and mortality in eNOFF patients, overall, there is a lack of consensus. Through this study, we aim to review the practice of blood transfusion and explore the possible association between blood transfusion and LOHS and short and long-term mortality in eNOFF patients.This was a retrospective study conducted in the Wrexham Maelor Hospital located in Wrexham, Wales, a part of the Betsi Cadwaladr University Health Board (BCUHB). It included all consecutive patients admitted to the hospital under the care of the Department of Trauma and Orthopaedics who were diagnosed to have eNOFF between January 2019 and December 2021. The included patients were >/= 65 years of age presentation. The fracture patterns included were intracapsular, extracapsular, and subtrochanteric femur fractures. Only the patients who required surgical intervention  were included in the study. eNOFF patients who were managed non-operatively were excluded from the study. Patients who were diagnosed to have pathological eNOFF or who suffered eNOFF because of poly-trauma and suffered additional fractures were also excluded.The demographic, medical history, operative, in-hospital treatment, and post-discharge details of the patients were accumulated from different online sources including the National Hip Fracture Database (NHFD), the transfusion lab information portal (Welsh Laboratory Information Management System (LIMS)), and the Welsh Clinical Portal. The information regarding the mortality of the patients was collected from the clinical information portal. We also used the actual patient notes from the hospital records section to add to any missing information. These resources are reliable clinical information portals for the patients treated under BCUHB. The requirement for blood transfusion was judged based on the patient's vital parameters, symptoms, and level of hemoglobin in the peri-operative period. This was also agreed upon by the treating orthogeriatrician in the ward-based multi-disciplinary team discussion.Statistical analysis was performed using IBM SPSS Statistics for Windows, Version 25.0 . Mean, median, standard deviation (SD), maximum, and minimum values, as well as the 95% confidence interval (CI), were calculated for each variable. In addition, we used the Student unpaired t-test and log-rank (Mantel-Cox) test for independent samples to determine significant differences between the groups of patients who did and did not receive a blood transfusion. For all tests, P < .05 was considered significant.Over the study period, a total of 501 eNOFF cases constituted the primary cohort of the study, with a mean age of 81 years (ranging from 65 to 102 years). Among the eNOFF cases, female patients (n=340) outnumbered male patients (n=161). Of the total cohort, 79 (15.8%) patients received a blood transfusion in the peri-operative period. While a higher proportion of female patients received peri-operative blood transfusions compared to male patients (61 out of 340 versus 18 out of 161), the difference was not statistically significant (p=0.07). Most of the transfused patients (52.9%) belonged to the American Society of Anesthesiologists (ASA) III category, but the need for blood transfusion did not differ significantly across ASA I, II, III, and IV categories (p=0.44) (Table The mean time to surgery was significantly longer in the transfused group (35.8 hours) compared to the non-transfused group 31.2 hours) (p=0.035). The average LOHS was significantly longer in the transfused group (22 days) compared to the non-transfused group (16.9 days) (p=0.022). The study population consisted of a considerable proportion of elderly patients, with 68% of the patients being in the higher subset of the elderly population. Additionally, a substantial proportion of the patients had a higher ASA grade, indicating a higher degree of co-morbidities. The ASA grade is a commonly used surrogate marker for assessing the preoperative co-morbidities index of patients. This indicates that the study cohort was representative of a population with significant preoperative risk factors that could potentially impact surgical outcomes  died during the five-year post-operative follow-up period. The early post-operative 30-day mortality rate was lower in patients who received blood transfusion (4%) compared to those who did not receive blood transfusion (9%). However, this difference in mortality was not statistically significant (p=0.07). At one year post-surgery, mortality was higher in the transfused group (33%) compared to the non-transfused group (26%)  compared to the non-transfused group (57.5%) (Figure .2% compaThe findings of this study support the practice of peri-operative blood transfusion in patients with low hemoglobin at presentation or in the early postoperative period as this reduces early postoperative mortality. The practice of blood transfusion does not cause any long-term deleterious effects or alterations in the physiology of the patient as the one-year and long-term five-year mortality rates are comparable for the eNOFF patients who did/did not receive a peri-operative blood transfusion. This positive effect on early postoperative mortality was also associated with statistically significant but marginally increased time to surgery and moderately increased post-operative LOHS.The population that suffers from eNOFF is usually medically compromised and suffers from one or more medical co-morbidities. These patients also tend to have poor physiological reserves. The outcome of the management of these patients is very dependent on the medical co-morbidities that they suffer from. This also influences healthcare costs because of increased medical support and delayed rehabilitation .One of the common peri-operative medical conditions in eNOFF patients is anemia. This could be pre-existing chronic nutritional anemia or because of blood loss from fracture or surgery. Anaemia in eNOFF patients influences the time to surgery, early postoperative recovery and rehabilitation, in-hospital stay, and healthcare costs . If not The incidence of blood transfusion in eNOFF patients in our series was 15.8%. This was much less than the reported incidence of blood transfusion in a nationwide study from the Republic of Korea, which is about 74% . In thisMortality in eNOFF patients is a well-studied outcome. Though the figures vary across studies, the national 30-day mortality following eNOFF, as reported in the NHFD 2021 report is 8.3% ,17. It cBlood transfusion is beneficial for the eNOFF patients when it is clinically indicated and, in the early postoperative period, the one-month mortality is less than the no blood transfusion cohort. Though this difference is not statistically significant, there is a clear trend of the benefit of blood transfusion as most of the patients who needed blood transfusion belonged to the high-risk group and belonged to the ASA III category. The long-term one-year and five-year mortalities were comparable in eNOFF patients who did or did not receive a blood transfusion. Thus, blood transfusion does not cause any deleterious long-term effects on eNOFF patients.LimitationsOur study had several limitations, including its retrospective design conducted at a single hospital, which may limit the generalizability of our findings. Furthermore, we did not conduct detailed analyses of individual co-morbidities in eNOFF patients who required blood transfusions. While receiving a blood transfusion was found to be associated with a longer time to surgery and LOHS, our study groups were not propensity-matched and statistical significance was not adjusted for observed differences. Therefore, it is important to exercise caution when attributing these outcomes solely to blood transfusion. Further research is needed to better evaluate the potential impact of confounding factors on these outcomes.Blood transfusion in eNOFF patients is a contentious issue. While it may be beneficial in reducing acute peri-operative mortality rates in high-risk patients, its impact on long-term mortality rates is inconclusive. Administering blood transfusion to eNOFF patients should be based on careful clinical assessment of the potential risks and benefits. Although blood transfusion can stabilize Hb levels and improve physiological reserves in high-risk patients, it also carries potential complications, including transfusion reactions and infections.Blood transfusion should not be considered a definitive solution for improving long-term outcomes in eNOFF patients, and its use should be carefully balanced against potential risks and benefits, with close monitoring and follow-up of patients being crucial for optimizing clinical outcomes.


Swab pooling may allow for more efficient use of point-of-care assays for SARS-CoV-2 detection in settings where widespread testing is warranted, but the effects of pooling on assay performance are not well described.We tested the Thermo-Fisher Accula rapid point-of-care RT-PCR platform with contrived pooled nasal swab specimens.We observed a higher limit of detection of 3,750 copies/swab in pooled specimens compared to 2,250 copies/swab in individual specimens. Assay performance appeared worse in a specimen with visible nasal mucous and debris, although performance was improved when using a standard laboratory mechanical pipette compared to the transfer pipette included in the assay kit.Clinicians and public health officials overseeing mass testing efforts must understand limitations and benefits of swab or sample pooling, including reduced assay performance from pooled specimens. We conclude that the Accula RT-PCR platform remains an attractive candidate assay for pooling strategies owing to the superior analytical sensitivity compared to most home use and point-of-care tests despite the inhibitory effects of pooled specimens we characterized. This glThe availability of new diagnostic tools has enabled public health efforts involving novel testing strategies that maximize limited resources and optimize public safety. Pooling allows for testing large groups of people while minimizing the consumption of limited resources, and is appropriate for settings where prevalence of the infection is sufficiently low that most individuals will test negative . PoolingThe Thermo-Fisher Accula is a rapid PoC platform for RT-PCR that has been FDA-approved for detecting influenza A and B, respiratory syncytial virus, and SARS-CoV-2 . The AccTesting pooled samples may introduce challenges not encountered with individual testing. Chief among these is the potential for compounding interfering substances in the pooling scenario. Consider nasopharyngeal sampling, where a swab is inserted into the nares or nasopharynx and then eluted into a liquid buffer. When a pooling strategy involves eluting swabs obtained from multiple individuals into a shared buffer, nasal mucous, inhaled medications, and other contaminants would be contributed from everyone in the pool, which may impact assay performance through additive effects. Further, a single inhibitor introduced from just one member of the pool would compromise the entire pool and, particularly if the result is falsely negative, undermine the pooling strategy.In this report, we describe the performance of the Thermo-Fisher Accula RT-PCR for contrived pools of six nasal swab specimens. We observe a reduction in assay performance which we postulate is mainly due to the presence of nasal mucous and debris. Although this appears to reduce the assay’s sensitivity, it is still an attractive candidate for PoC pooling strategies owing to the superior sensitivity of RT-PCR compared to antigen tests.2.2.1.All Accula experiments were performed with the following reagent deposited by the Centers for Disease Control and Prevention and obtained through BEI Resources, NIAID, NIH: Quantitative PCR (qPCR) Extraction Control from Heat-Inactivated SARS-Related Coronavirus 2, Isolate United States-WA1/2020, NR-52350, lot 70036662.2.2.The FLOQSwabs® 502CS01 Regular Size Nylon® Flocked Swab  was used for experiments and collection of Negative Nasal Matrix.2.3.Negative matrix specimens were prepared from volunteers recruited at Thermo Fisher, Emory University, or community testing sites in the metro Atlanta area following provision of informed consent under protocols approved by the institutional review boards of Mesa/Thermo Fisher or Emory University (STUDY00001082) and in accordance with relevant guidelines and regulations. Active recruitment at Emory University and metro Atlanta testing sites occurred between July 6, 2020 and October 12, 2021. The authors did not have access to identifying information during or after experiments. Volunteers contributing to the negative matrix performed self-swabbing of the anterior nares from both nostrils according to the Accula instructions for use (IFU) . Pooled 2.4.PNM and INM limit of detection (LOD) studies followed a protocol similar to those used in recent US Food and Drug Administration (FDA) Emergency Use Authorization filings in which 50 μL of diluted NR-52350 was placed on a dry swab prior to introducing the swab into the PNM or INM . An alte2.5.4 Results for all experiments were observed within 15 min of assay completion.The Accula RT-PCR assay consists of a single-use cassette and a multiple-use reader . All ass2.6.Two dry swabs were spiked with 50 μL of NSB containing 250, 750, 1,500, 2,250, 3,000, 3,750, or 4,500 copies of NR-52350. One swab for each copy number was then eluted into one PNM, and its pair was eluted into one INM specimen and subsequently stored on ice. Three assays were performed on each specimen. The lowest copy number specimen with 3 of 3 positive results was then used to perform 20 additional assays. If fewer than 19 of these assays were positive, 20 assays were performed from the next highest copy number specimen. This was repeated until at least 19 of 20 assays were positive.2.7.For swab sequence testing, six asymptomatic volunteers were provided with three swabs each. To minimize the potential confounding by repetitive swabbing, collection was performed in the following sequence: Swab A was inserted into the left nostril and the circumference of the anterior nares was brushed 10 times in accordance with the Accula IFU. Next, swab B was inserted into the right nostril with brushing. Then, the right nostril was brushed with swab A followed by brushing of the left nostril with swab B. Finally, swab C was collected by brushing both nostrils without specification of which nostril should be swabbed first.Swab C from each of the six volunteers was used to perform an individual test to verify the volunteer was negative for SARS-CoV-2. Two pools were created from swabs A and B from the six volunteers . Pool I 2.8.To aid in the interpretation of the LOD determined by pooling experiments with the Accula in the context of other PoC and home-use tests, we referenced data generated at our institution from LOD experiments performed with several common SARS-CoV-2 antigen assays and the LOD claimed in FDA EUA submission data for CLIA-waived PoC molecular assays.8 genome equivalents/mL) was used simultaneously as a calibration from which RNA copy number per mL was calculated. A standard curve for the N2 gene primers was generated by diluting the synthetic RNA 10-fold and amplified by quantitative RT-PCR reaction.The LOD experiments for antigen tests were performed by preparing a panel of dilutions using pooled remnant clinical samples with SARS-CoV-2 lineage B.1.2 in nasal wash or nasal swab matrix according to the matrix most compatible with each assay. The claimed LOD for molecular tests was determined by referencing the IFU published on the FDA website. 3.3.1.Initial assays produced 3 of 3 positive results from INM specimens eluted with 750 copies and greater. Twenty-replicate reflex testing of identical specimens produced 14 positive, 5 negative, and 1 invalid result from both 750 and 1,500 copy specimens . The 2,23.2.Twenty assays were performed from two newly prepared, never-frozen PNM from six volunteers. The PNM specimen prepared by spiking the first of six swabs produced 19 positive and 1 invalid result . The PNM3.3.Five PNM specimens (A-E) were spiked with heat-inactivated SARS-CoV-2 (BEI cat# NR-52350: United States-WA1/2020) to an approximate concentration of 100 genome copies per mL. Specimen B was noted to be more turbid than the other four specimens with visible debris . Ten ass3.4.To examine potential deleterious effects of the TP independent of nasal mucous and debris, NR-52350 was diluted in NSB to approximately 100 cp/mL concentration. 20 assays were performed under the following conditions: (i) TP specimen loading, (ii) MP specimen loading and (iii) transfer of specimen into a secondary container with the TP followed by loading of the transferred sample into the assay cassette with the MP . Results3.5.LOD testing experiments from 8 antigen-based SARS-CoV-2 assays performed using individual nasal matrix compatible with each test suggested that none of the common assays exhibited a lower LOD than the Accula platform with PNM . The meaWe also compared our results to the claimed LOD for molecular tests with EUA as listed on the FDA website . These a4.Strategies for promoting the safe gathering of groups of people in workplaces, schools and at large events has varied across the course of the COVID-19 pandemic. While individuals with symptoms of illness should always be encouraged to avoid others, the potential to spread SARS-CoV-2 exists in individuals with subclinical or absent symptoms. To minimize risk, some programs and institutions have required proof of vaccination or even employed active testing programs. When a testing strategy is desired, various approaches can be considered. Individual testing with home use diagnostic tests may be prohibitively costly and produce significant waste, and many home use tests suffer from inferior sensitivity. The swab pooling strategy promises more efficient use of testing resources while allowing for use of more sophisticated (and likely more sensitive) diagnostic instruments, yet increased test turnaround time and the added expense of transporting specimens may prohibit swab pooling in a centralized laboratory.Swab pooling with a PoC instrument may uniquely meet the needs of many events or institutions by allowing pooled testing in the proximity of a large gathering. However, the impact of any pooling strategy for PoC SARS-CoV-2 diagnostics has only been described in a few studies  and to oWe hypothesized that impaired assay performance of pooled specimens could be attributed to an overall greater burden of nasal mucous and debris as swabs from multiple individuals are eluted into the same volume of buffer designed for individual testing. The observation of fewer false negative and invalid results when loading PNM specimens at 100 copies/mL (slightly below the LOD reported in the Accula EUA) with an MP compared to a TP has led us to postulate that the smaller bore of the MP may filter some particulate or produce shear forces that achieve greater sample homogenization and reduce the burden of these inhibitors in the specimen. TP-loaded specimens with 300 copies/mL appeared to overcome this problem, suggesting this inhibition is not as significant at higher target concentrations. We observed that the sample with distinctly greater turbidity and particulate contents uniquely performed worse despite the higher target concentration, suggesting that the deleterious effect of nasal mucous and debris likely also depends on the amount of the nasal mucous. These observations indicate that pooling strategies for PoC assays must consider the compounding of nasal mucous and debris. For example, anticipatory guidance in pooling IFU could warn the assay operator that visibly turbid pooled specimens may warrant re-testing of individuals separately to avoid the undesirable effects of a single individual who contributes high quantities of mucous.Our experiments in buffer with intermediate transfer of specimen using the TP rule out a component of leeching chemical inhibitor from the TP plastic material or binding of viruses or viral RNA to the TP. However, the observation that the TP-loaded specimens performed worse than the MP-loaded specimens (with or without intermediate transfer using the TP) suggests that there is also a disruptive effect from the TP independent of the presence of nasal mucous. We speculated that this might be due to the TP’s introduction of air bubbles to the microfluidic cartridge, which could inhibit the assay. This phenomenon warrants further investigation to weigh the negative effects of the TP on assay performance against the need for a single-use, fixed-volume tool that may not provide the precision of a standard laboratory micropipette.Our observations of the effects of pooling on the performance of the Accula assay are limited by use of contrived specimens but are expected to simulate real-world pooling conditions. The experiments in To provide context for the interpretation of our measurements with the Accula system, one must consider the available options when screening a large group of people for SARS-CoV-2 infection. One strategy is to have all individuals at an event or gathering individually perform a test with home-use approval without the need for a certified laboratory space. This strategy is resource-intensive (one test is required for every participant) and the expected LOD is similar to those with home-use approval presented in An alternative approach is to perform pooling with a test that has been assigned a CLIA-waiver. This requires certification of a laboratory space by the Centers for Medicare and Medicaid Services (CMS) or approved accreditation agency, which may be appropriate for venues  with compatible facilities . The dat5.In summary, our data suggest that specimen pooling may have an inhibitory effect on LOD of the Accula SARS-CoV-2 RT-PCR assay and that this is likely due to a more significant burden of nasal mucous and debris in the pooled specimens. However, pooled PoC RT-PCR on the Accula platform still exhibits better LOD compared to individual use of most common FDA-approved home tests with a format compatible with swab pooling, and therefore warrants consideration in pooling strategies. Limitations of our work include the use of contrived specimens with heat inactivated virus and use of the United States-WA1/2020 isolate which is no longer representative of currently circulating variants, although assay performance of the Accula is not thought to be significantly affected by mutations in newer variants to date . FurtherThe original contributions presented in the study are included in the article/The studies involving humans were approved by Emory University Institutional Review Board. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written and/or verbal informed consent to participate in this study.ML, KF, and GD performed the Accula experiments. JS, WO, KM, HW, and JR supported the experiments and/or assisted with specimen collection, handling and storage. AR, LB, and MG designed and/or performed the antigen test LOD studies. PM, WK, ET, WL, ML, KF, and GD contributed to the design and interpretation of Accula experiments. All authors contributed to the article and approved the submitted version.This work was supported by NIH grant U54EB027690 to WL and NIH grant UL1TR002378.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


COVID-19 typically presents with flu-like symptoms due to the viral infection itself. The most severe cases are characterised by lung damage, an important factor in fatal outcome due to alveolar damage. In some cases, patients develop a long COVID with persistent symptoms of chest pain and fatigue. Causes, including organ damage or inflammation, are being investigated. Clinical outcomes are variable and permanent lung damage is not fully understood, while vaccination is effective against severe infection but its effect on respiratory function in mild cases remains uncertain. This retrospective study aims to analyse changes in lung function in HCWs who had COVID-19 between 2020 and 2022, comparing their spirometric test results before and after the pandemic and taking into account their vaccination status. 321 HCWs were included in the study. The study examined spirometric parameters both before and after the pandemic, and all measured outcomes except the FEV1/FVC ratio showed a significant decrease during the study period. We then assessed the association between SARS-CoV-2 infection and changes in lung function parameters, analysing infections in 2020, 2021 and 2022 separately. We found a statistically significant difference in Forced vital capacity (FVC) between infected and non-infected subjects in 2020 and 2021, but not in 2022. To evaluate the protective effect of SARS-CoV-2 vaccination on respiratory function, a linear regression analysis was performed using changes in FVC, Forced expiratory volume in 1 s (FEV1), FVC/FEV1 ratio and Peak expiratory flow (PEF) as dependent variables. The analysis showed that the decline in FVC was significantly lower in subjects who had been vaccinated prior to infection. The study concludes that subclinical SARS-CoV-2 infections in 2020 and 2021 worsened respiratory parameters (FVC and FEV1), but vaccination protected against these effects. Even healthy individuals with previous infections showed respiratory changes, with vaccination providing protection, especially for FVC decline. This highlights the importance of vaccinating healthcare workers against COVID-19. Typically, COVID-19 presents with flu-like symptoms that are due to the viral infection itself. Severe cases of COVID-19 are mainly characterised by lung damage, which is the main cause of fatal outcome due to severe alveolar damage. This damage differs from classical respiratory distress syndrome (ARDS) by the absence of symptoms proportional to the severity of hypoxaemia . Gross eIn this study, we aimed to assess any variation in pulmonary function parameters in a population of healthcare workers (HCWs) who experienced COVID-19 during the period 2020–2022 by comparing spirometric tests performed before and after the SARS-CoV-2 pandemic, in relation to their vaccination status.In this study we retrospectively collected lung function tests of HCWs who underwent their health surveillance visit at the Policlinic of Rome Tor Vergata in the period 2019–2023.At the policlinic, HCWs undergo an annual spirometric test to assess their fitness for work. In accordance with the recommendations of the Italian Ministry of Health, routine pulmonary function tests were suspended during the SARS-CoV-2 pandemic (from March 2020 to September 2022) due to the high risk of SARS-CoV-2 transmission.To assess any changes in respiratory function that could be attributed to SARS-CoV-2 infection, we compared spirometry tests collected in the six months prior to March 2020 with those performed by the same operators in the six months following September 2022.Data on SARS-CoV-2 infections were collected from the occupational health database. During the pandemic, each hospital HCWs had to undergo a nasopharyngeal swab every 15 days, and the results of these tests were collected in a unique database. In addition, symptomatic SARS-CoV-2 infections occurring in those operators were routinely reported to the occupational health department and recorded in the same database. Thus, data on both symptomatic and asymptomatic COVID-19 were available for all hospital workers. SARS-CoV-2 vaccination history was extracted by the regional vaccine database that includes all residents who received the Sars-CoV-2 vaccine in the period 2020–2023.Spirometric test was conducted accordind to ERS/ATS standards . Forced HCWs had to meet the following criteria to be included in the study: having had a valid pulmonary function test both before and after the pandemic (in the previously described defined period), absence of significant comorbidities, mainly respiratory diseases, availability of results from molecular swab tests performed during the pandemic period, availability of documentation on vaccination status. Subjects with severe infection were excluded from the study .The following parameters were collected for each partecipant: gender, age, weight and height (BMI), smoking habit, comobridities, COVID-19 (number of episodes and date), and vaccination for SARS-CoV-2 (number of doses). Results of spirometric tests both in the pre- and in the post-pandemic period were also collected and the following paramethers were recorded: FVC, FEV1, Tiffenau index and PEF. All spirometry was recorded as a measured value (lt for volumes and l/sec for flows) and as a percentage of the predicted value.Data were entered into an Excel database for subsequent analysis. Mean, standard deviation, range, and distribution were calculated for continuous variables. Categorical variables were expressed in terms of frequency.A paired sample t-test analysis were conducted to analyze spirometric parameters in the pre- and in the post-pandemic period. A linear regression was also used to compare the year of disease onset with each spirometric parameter.Subsequently, linear regression was performed between the examined variables  and the investigated factors  for multivariate analysis.For association tests with the listed variables, a significance level of 5% (p < 0.05) was required.We analyzed data from 321 HCWs. Five subjects were excluded from the study. One had a severe infection and the other four had respiratory comorbidities (asthma and COPD). Mean age was 48,85 ± 10,63 (range 20–74) years. Regarding COVID-19, 55.1% of the subjects (177) had a recognized infection in the examined period. Concerning vaccination history, 95.0% (305) of the study population received the vaccination for SARS-CoV-2, and of these 40.7% (124) developed a breakthrought infection after receiving at least one dose of vaccine. The main characteristics of the study population are shown in Table Table Spirometric paramethers were analyzed both in the pre- and in the post-pandemic period. All collected outcome, except FEV1/FVC ratio showed a significant reduction during the study period when tested at paired-samples t-test Table .Subsequently, we tested the correlation between the COVID-19 and the worsening of each lung function parameter. As the different strains of SARS-CoV-2 showed different pathogenicity in the upper and lower respiratory tract, we analyzed the influence of infections in the three study years  separately. Linear regression showed a statistically significant difference in FVC between infected and non-infected subjects in 2020 and 2021, but no significant difference in 2022, as shown in Table To assess the protective effect of SARS-CoV-2 vaccination on respiratory function, we performed a linear regression between the examined variables  and the investigated factors  for multivariate analysis. The decline in FVC was significantly lower in subjects who had received vaccination prior to infection. Results are shown in Table It is now more than evident that COVID-19 is a complex disease, capable of manifesting itself in a rather varied manner in the population, affecting many organs and systems with varying degrees of severity. The importance of our study lies in the inclusion of individuals without significant comorbidities who showed a statistically significant decline in lung function indices  during follow-up spirometry, which persisted over the medium to long term.Our study revealed subclinical changes in the lung function of the subjects included in the sample. Since a physiological decline in lung function indices is expected over a three-year period, in order to highlight the role of COVID-19 infection, we compared the loss of FEV1, FVC and FEV1/FVC in the group of subjects who had the infection in comparison to those who did not become infected. Although no significant differences were found between the two groups overall when considering COVID-19 infection as a whole, the analysis of the different years showed that infection with in 2020 and 2021 (time of spread of the first variant strains) was associated with a slight but significant reduction in FVC. Previous studies had already found a reduction in lung function indices in patients with COVID-19. Referring to another international study, the COMMUNITY study assessed long-term immunity in healthcare workers at a Swedish hospital who had a mild form of the disease during the acute phase . The stuTo test the hypothesis that previous vaccination with Comirnaty (BNT162b2) is protective with respect to worsening lung function indices, we entered this vaccination into a multiple regression model, considering age, gender, BMI and smoking as covariates. Vaccination was statistically associated with a lower loss of FVC emphasising the importance of vaccination in protecting even from subclinical effects of COVID-19 infection.It is necessary to specify that our investigation was primarily limited by the relatively small sample size (321 operators), which could have led to falsely negative results in determining additional variables of functional loss. However, given the correlation between SARS-CoV-2 positivity and changes in functional indices, it is reasonable to assume that this result was not influenced by experimental limitations.There is a notable heterogeneity in the number of COVID-19 cases that developed within our sample over different years. This is another limitation of our study, attributable to the epidemiological characteristics of the infection.Certainly, further studies are required to extend the follow-up period, allowing for a better evaluation of the significance of the reductions in spirometric indices recorded by us, which are still in the subclinical phase. As a result, the prognostic significance of our analysis results remains limited.Undoubtedly, the evidence of unfavorable functional outcomes in a population represented by subjects without risk factors and with a history of mild disease can contribute to increasing the perception of COVID-19-related risk. The lack of risk perception resulting from the disease has been responsible for a significant increase in vaccine hesitancy, even among healthcare workers.Despite the aforementioned limitations, our work demonstrates the effectiveness of the vaccination campaign in preventing unfavorable outcomes on functional indices, a factor to consider in defining vaccination strategies for populations at higher risk of infection, such as healthcare workers.The results of our study shed important light on the impact of subclinical infection in 2020 and 2021, which coincides with the circulation of the alpha, beta and delta variants of SARS-CoV-2. Notably, we observed that such subclinical infections were associated with a deterioration of key respiratory parameters, specifically FVC and FEV1. This suggests that even mild or asymptomatic infections during the delta variant-dominated period could have significant implications for respiratory health.On a positive note, our research also revealed a promising trend concerning vaccination. We found that individuals who received vaccination appeared to be better protected against the adverse respiratory outcomes associated with subclinical infections. Even in healthy subjects who had experienced an infection, the presence of vaccination seemed to exert a protective effect on respiratory function, potentially mitigating the impact of the infection on lung health. In the literature, we can see that vaccination is associated with a reduction in inflammation in the short and long term, which could partly explain the reduced impact of COVID-19 on lung function parameters in vaccinated subjects .These findings underscore the crucial importance of widespread vaccination, especially among healthcare workers. As frontline heroes battling the COVID-19 pandemic, their health and well-being are of paramount importance. By ensuring universal vaccination of healthcare workers against COVID-19, we can take proactive measures to safeguard their respiratory health and reduce the risk of potential long-term consequences resulting from subclinical infections.In summary, our study not only emphasizes the significance of addressing subclinical infections during the delta variant surge but also highlights the encouraging role of vaccination in mitigating the adverse respiratory effects of such infections. Implementing universal vaccination among healthcare workers is a vital step towards safeguarding their health and the well-being of the broader community in the ongoing fight against the COVID-19 pandemic.In conclusion, our study shows that subclinical infection during the year 2020 and 2021 is associated with a worsening of respiratory parameters (FVC and FEV1), but vaccination appears to be associated with protection against these outcomes.In addition, it is highlighted that even in healthy subjects who have had an infection, respiratory outcomes can be detected, towards which vaccination appears to have a protective effect, shown in all lung function parameters, but particularly in the decrease in FVC. This evidence underlines the need for universal vaccination of healthcare workers against COVID-19.


This study demonstrated that computational modeling can provide valuable insights to improve the pre-procedural planning of TAVR-in-TAVR.Transcatheter aortic valve replacement (TAVR) is increasingly being considered for use in younger patients having longer life expectancy than those who were initially treated. The TAVR-in-TAVR procedure represents an appealing strategy to treat failed transcatheter heart valves (THV) likely occurring in young patients. However, the permanent displacement of first THV can potentially compromise the coronary access and ultimately inhibit the blood flow circulation. The objective of this study was to use finite-element analysis (FEA) to quantify coronary flow in a patient who underwent TAVR-in-TAVR. A parametric investigation was carried out to determine the impact of both the implantation depth and device size on coronary flow for several deployment configurations. The FEAs consisted of first delivering the SAPIEN 3 Ultra THV and then positioning the Evolut PRO device. Findings indicates that high implantation depth and device undersize of the second THV could significantly reduce coronary flow to 20% of its estimated level before TAVR. Additionally, a positive correlation was observed between coronary flow and the valve-to-coronary distance (R = 0.86 and  Transcatheter aortic valve replacement (TAVR) has now become the standard of care for elderly patients with aortic stenosis, as compared to conventional open-chest surgery . OngoingIn this setting, the deployment of a new THV in the failed one  is an attractive therapeutic option to further extend the life expectancy of TAVR patients. Although TAVR-in-TAVR is clinically feasible, the new THV permanently displaces the valve leaflets of the first device towards the metallic stent frame by generating a tube graft that extends from the annulus to the sinus of Valsalva or above the sino-tubular junction. This not only impairs coronary access but also compromises coronary flow. Using computed tomography (CT), Forrestal and colleagues  demonstrWith this in mind, we conducted a study to determine the flow of blood circulating in coronary arteries under different implantation configurations and device sizes using patient-specific computational analysis of TAVR-in-TAVR. Specifically, we investigated a patient with a degenerated SAPIEN 3 Ultra THV that was treated with the self-expanding Evolut Pro during a second treatment. By simulating the deployment of each device and performing smoothed-particle hydrodynamic (SPH) analysis, we not only evaluated the feasibility of TAVR-in-TAVR, but also gained a better understanding of how hemodynamic performance varies with different deployment configurations.3. After 3 years from TAVR, early device degeneration characterized by valve thrombosis and leaflets thickening was observed during echocardiography. Specifically, hypo-attenuated leaflet thickening of SAPIEN 3 Ultra THV with reduced leaflet motion was observed. Based on the surgical risk and patient clinical history, the Heart Team decided to implant a 29-mm Evolut PRO device through TAVR-in-TAVR to treat the degenerated bioprosthesis. A strict ECG-gated CT imaging was performed to evaluate the stenotic aortic valve before and after each device implantation. Therefore, a total of three ECG-gated CT studies were conducted for this patient:• The first was to evaluate the aortic valve annulus of the diseased valve .• The second aimed to assess the functionality of the initially implanted bioprosthesis .• The third was conducted to evaluate the deployment of the second bioprosthesis .We investigated the case of a 68-years-old gentleman with severe aortic stenosis, which was treated with a 23-mm SAPIEN 3 Ultra THV. Baseline patient characteristics were: heartbeat of 65 bpm, systolic blood pressure of 113 mmHg, diastolic pressure of 65 mmHg, peak gradient of the stenotic valve of 60 mmHg, transaortic flow jet of 3.9 m/s, aortic valve annulus of 22.8 × 23.0 mm and calcium volume of 1,387.4 mmECG-gated CT images of the patient prior to TAVR were processed in Mimics  to reconstruct the geometry of the aortic root. This involved semi-automatic thresholding of contrast-enhanced images, followed by manual editing and smoothing . CalcifiEach anatomic part was meshed using ICEM meshing software  after convergence analysis. We used the stress parameter  to calculate the discretization errors and estimates were kept <5%. Thus, the patient-specific model was meshed with unstructured triangular shell elements for the aortic root surface (with a size of 0.6 mm) and tetrahedral solid elements for calcifications (with a size of 0.5 mm). Native valve leaflets were initially meshed with triangular shell elements and grid size of 0.12 mm, which were then protruded to generate a solid part with four layers of elements along the thickness direction. 2 at CT scan. We assumed a Neo-Hookean material model for the material behavior of both the aortic root and native valve leaflets. Calcifications were embedded in the native valve leaflets and had a linear-elastic material behavior (E = 400 MPa and n = 0.47)  . The invThe structural heart intervention was simulated using finite-element analysis (FEA) with Abaqus/Explicit software . To model the dynamic phenomenon, we used a quasi-static FEA approach that ensured the kinetic to internal energy ratio remained below 10%. This was achieved through a mass scaling technique using a stable time increment and thus reducing the computational cost. In addition, a penalty contact algorithm with a friction coefficient of zero was used to enable contact during simulations.Bioprosthesis models were meshed with structured hexahedral elements, as reported in previous studies by our group see  Pasta e. StainleCrimping of SAPIEN 3 under frictionless contact conditions was carried out using a cylindrical surface gradually moved along the radial direction from the initial device diameter of 23 mm to the final diameter of 6 mm at the device crimped stage. Then, the SAPIEN 3 Ultra device was placed in the patient-specific model with an implantation depth of 5 mm whilst a restart analysis was considered to account for the stress state resulting from the crimping numerical analysis. After crimping, an elastic recoil of SAPIEN 3 Ultra was observed. For the sake of simplicity, expansion of SAPIEN 3 Ultra THV was performed by radially displacing a cylindrical surface representing the wall of the expanding balloon. Expansion was obtained by enlarging the balloon surface upon the device nominal diameter of 23 mm. This can be considered a valid approach since angiography usually highlights negligible axis rotation and translation during device expansion.After deploying the SAPIEN 3 Ultra, the Evolut Pro was positioned in the post-TAVR model with an implantation depth of 9 mm and was then crimped with an approach similar to that of first device. By pulling the sleeve towards the distal ascending aorta and releasing the device, the Evolut Pro stent was gradually deployed into the first device to mimic the TAVR-in-TAVR.• 5-mm implantation depth for the 23-mm SAPIEN 3 Ultra and 9-mm implantation depth for the 29-mm Evolut Pro —reference model as clinically done by the Heart Team.• 5-mm implantation depth for the 23-mm SAPIEN 3 Ultra and 16-mm implantation depth for 29-mm Evolut Pro —low TAVR-in-TAVR implantation• 2-mm implantation depth for the 23-mm SAPIEN 3 Ultra and 9-mm implantation depth for the 29-mm Evolut Pro —high TAVR implantation• 5-mm implantation depth for the 23-mm SAPIEN 3 Ultra and 9-mm implantation depth for the 26-mm Evolut Pro —device undersizing of TAVR-in-TAVRA parametric analysis of the TAVR-in-TAVR procedure was carried out by varying the implantation depth and device size with respect to the baseline model. The following scenarios were therefore explored:After the TAVR-in-TAVR simulation, the smoothed-particle hydrodynamics (SPH) modeling approach was used to quantify flow circulating in coronary arteries. The SPH approach, adopted in previous studies , is usefTo compare CT images of TAVR-in-TAVR with FEA predictions, the device expansion was computed using the nominal prosthesis size as a follow  × 100. Specifically, measurements were taken at three cross-sectional level including the inflow, mid and outflow of TAVR-in-TAVR. The valve-to-coronary (VTC) distance was measured in the short-axis view after multiplanar reformations as the length from the metallic device frame to the ostia of left coronary artery (LCA) and right coronary artery (RCA). The valve-to-coronary (VTC) distance was measured in the short-axis view as the length from the metallic device frame to the ostia of left coronary artery (LCA) and right coronary artery (RCA). The coronary flow percent was computed as the ratio of the coronary flow for each TAVR-in-TAVR scenario divided by the flow of the model prior to TAVR-in-TAVR. The linear relationship between coronary flow percent and VTC distance was investigated by Pearson correlation.Several steps of the deployment of both SAPIEN 3 Ultra and Evolut Pro THVs are shown for the axial and sagittal views of the patient anatomy . After cA qualitative comparison was performed between FEA and imaging of TAVR-in-TAVR for the reference S3-in-EV model, which mimicked the implantation depth performed by the Heart Team . The expThe flow velocities over the cardiac beat were analyzed to investigate the flow circulating in the coronary arteries after TAVR-in-TAVR . During p = 0.032), while the correlation between VTC distance and RCA coronary flow was stronger (R = 0.93 and p = 0.014).In this study, computational modeling was used to investigate the impact of TAVR-in-TAVR on coronary flow as the placement of the second prosthetic valve in the degenerated device can cause changes in the hemodynamics of the aortic root. The parametric analysis we conducted revealed that variations in the implantation depth or device size can alter the flow circulation compared to pre-TAVR flow analysis. We also discovered that the VTC distance, used as a pre-procedural metric for patient risk stratification, correlates with the amount of flow circulating in the coronary arteries. While this study is limited to this specific patient case, the identification of design and positioning strategies that optimize coronary flow has the potential to enhance the clinical outcomes of TAVR-in-TAVR procedures and mitigate the risk of adverse events.Coronary artery obstruction is four times more common for valve-in-valve deployment when compared to traditional TAVR, as the leaflets of the degenerated THV become tilted vertically by the second device, creating a cylindrical covered stent frame . Thus, TSeveral computational studies have examined the effects of various procedural factors on TAVR configuration, including the implantation depth and tilt angle , the utiTo avoid numerical issues due to large displacements and complex contact conditions, the SAPIEN 3 Ultra valve leaflets were not simulated in this study. This may have altered the resulting coronary flow estimates; although, the covering of whole stent frame with a surface has most likely resulted in the analysis of the worst case scenario. Most importantly, the SPH method is limited by the particle size and not any verification on the mesh sensitivity was carried out in consideration of the small caliber of coronary vessels. Furthermore, the distal ends of each outlet were closed off with a surface to prevent particle dispersion outside the solid domain. This could have affected the blood flow conditions at the boundary, despite the outlets being adequately extended. This can impact the blood flow conditions at the boundary, though the outlets were sufficiently extended. A Neo-Hookean material model was used for the aortic root model, despite the fact that the soft tissue is hyperelastic and anisotropic materials. A Neo-Hookean material model was used for the aortic root model and the device valve leaflets, despite the fact that the soft tissue is hyperelastic and anisotropic materials. However, imaging data of patient undergoing TAVI suggests stiffening of the aortic wall with very low circumferential strains due to patient’s advanced age. Our assumption aligns with the findings of This study revealed important insights into the deployment of THVs when the first device experiences structural valve failure. The risk of device obstruction on the resulting coronary flow after TAVR-in-TAVR depends on both the original implant and the device to be implanted. By using computational models to simulate blood flow patterns and assess the impact of different deployment strategies on coronary flow, clinicians can better predict and manage the risk of coronary obstruction during TAVR-in-TAVR procedures. Future studies should continue to investigate the impact of different factors such as patient anatomy, implantation depth, and device size on coronary flow, especially in TAVR-in-TAVR.