An electroencephalography (EEG)-based brain–computer interface (BCI) is a system that provides a pathway between the brain and external devices by interpreting EEG. EEG-based BCI applications have initially been developed for medical purposes, with the aim of facilitating the return of patients to normal life. In addition to the initial aim, EEG-based BCI applications have also gained increasing significance in the non-medical domain, improving the life of healthy people, for instance, by making it more efficient, collaborative and helping develop themselves. The objective of this review is to give a systematic overview of the literature on EEG-based BCI applications from the period of 2009 until 2019. The systematic literature review has been prepared based on three databases PubMed, Web of Science and Scopus. This review was conducted following the PRISMA model. In this review, 202 publications were selected based on specific eligibility criteria. The distribution of the research between the medical and non-medical domain has been analyzed and further categorized into fields of research within the reviewed domains. In this review, the equipment used for gathering EEG data and signal processing methods have also been reviewed. Additionally, current challenges in the field and possibilities for the future have been analyzed. The electroencephalography (EEG)-based brain–computer interface (BCI) is one of the most rapidly developing fields of BCI ,2 and haThe BCI applications have been initially developed to help people with disabilities to be able to communicate, control computers and use aiding equipment such as wheelchairs or robotic arms. One of the first BCI applications assisted individuals with speech anomalies . The posEEG-based BCI applications have been used in different areas stretching from the medical domain to non-medical domain where EEG-based BCI applications have been used for entertainment, art, as well as some other areas ,6,13. ThDuring the current review, the literature on EEG-based BCI applications in the period from 2009 to 2019 was analyzed. The current review gives an overview concerning articles and conference proceedings per year, publications per region/continent, experimental publications per year, publication distribution by domain, EEG devices used, number of EEG channels used, techniques used to obtain EEG data, feature extraction and classification methods used.In the first part of the review, an overview concerning the published articles as well as conference papers is also presented through the years. The regions/continents at the forefront of the scientific progress in the field have been highlighted.An overall review of EEG-based BCI applications has been created and the BCI applications have been categorized based on domain  and by field, describing the current trends in BCI applications development in more detail. The BCI applications have been included in the medical domain in case they have been designed to provide assistance, monitor, assess or support rehabilitation and in the non-medical domain in case the applications have been designed to entertain, control machines, authenticate, monitor or assist without a medical purpose. The distinction between the domains has been done in a way similar to the work by Al-Nafjan et al. .This review introduces the current trends for the development of EEG-based BCI applications. Although the initial need for BCI applications has been in the medical domain, this review shows that there is currently a higher pace of development in the non-medical domain. The applications meant for widespread use could be used for monitoring user attention, supporting daily activities or for entertainment.In this review, EEG signal acquisition and processing is also analyzed. The overview concerning the prevalence of different EEG devices has been given together with the details regarding the number of EEG channels used in previous studies. An overview is given concerning the techniques used to obtain the EEG data. Regarding the EEG signal processing, feature extraction and classification methods have been reviewed.This review was conducted in connection to the first author’s master’s thesis . This reThe ethical, legal and safety considerations in BCI application development are as important as the technical aspects. The synthesis on current challenges gives a mapping of what to focus on in order to support BCI development and shows where the possible risks exist that would need to be addressed. The trends and future possibilities give a better understanding of what we could expect in years to come.BCI can be defined as a system that translates the brain activity patterns of a user into messages or commands for an interactive application . A BCI iAccording to Padfield et al. , the sigThe most common way to categorize BCI is on the basis of invasiveness. The BCI could be invasive or non-invasive, depending on whether the device is connected physically to one’s brain and the electrodes are placed inside the brain or the device will read brainwaves from the top of the scalp . The useMore popular and easy is the non-invasive method, where the brainwaves are read from the top of the scalp via electrodes located on the scalp. As this method also has some drawbacks, including disturbance from external noise, effects from the posture and mood of the subject, detecting a low strength signal resulting in a reduced signal quality , there iThe most popular current non-invasive method for acquiring brainwaves for BCI is detection through EEG. The popularity of EEG is supported by the inexpensiveness of the equipment, reduced complications compared to invasive procedures, portability, easy process to set-up and use and the possibility to directly measure neural activity ,2. EEG rBCI could be divided into separate subgroups based on the way to detect and convey the signal from the brain to BCI application and principles of the functioning of the BCI. As per Al-Nafjan et al. , BCIs coThe BCIs using active control react on conscious efforts to alter brainwave patterns and the applications could be controlled via active efforts by the user. The BCIs applying passive control react to the involuntary status of the brainwaves, for example on emotional states such as meditation, excitement and stress. Different emotions could be elicited by, for example, visual-based elicitation using images, prepared task or audio-visual elicitation using short film video clips .During active control of the BCI application, the signal could be detected via different techniques. The range of these techniques is broad and covers motor imagery, external stimulation , error-related potential, hybrid and other techniques. During the motor imagery task, for example, the subject is imagining the movement of a specific body part, and during error-related potential task, error-related potential is generated when there is a mismatch between the subject’s intentions and response from the BCI application .As per Pasqualotto et al.  and Mach“Dependent BCIs” require muscle control, for example, via gaze control. “Independent BCI” on the other hand detect signals only based on changes in the brainwaves without required muscle movement ,26. AccoAn additional way to classify BCIs is based on the time when the signals from the user are gathered by the BCI. The BCIs could therefore be divided as synchronous and asynchronous . BCI is The systematic literature review was prepared based on three databases, PubMed, Web of Science and Scopus, in order to cover a wide range of reliable peer reviewed publications on EEG-based BCI applications. In previous studies, either one  or multiThe time period for the publications from between 2009 and 2019 was selected in order to give an overview concerning longer period of time and to be able to compare the results with other studies. While 2019 might not seem truly up to date, we would like to highlight that it is a systematic review. Thus, once such a review has begun, altering the chosen period would mean either (i) compromising the systematic nature of the review, or (ii) conducting a new systematic review from scratch, which are undesirable and impractical, respectively. Similar lengths of the time interval have been selected in previous studies ,28. FromAfter the identification of the publications, a screening of the publications was performed. After the assessment of eligibility, 202 publications were included in the final analysis. The process was conducted as per the PRISMA model . Listed Determine the trends concerning the publication of articles, conference proceedings and overall number of publications on EEG-based BCI applications from 2009 until 2019.Determine the techniques used for obtaining EEG data and give an overview concerning the trends in EEG signal processing, which includes feature extraction and classification methods.Give an overview concerning the devices used for EEG signal collection and specify the number of EEG channels used for obtaining the data.Determine the proportion of scientific studies conducted on the topic in the medical and non-medical domain and further analyze the distribution of the studies per application field.Analyze the literature by regions/continents, i.e., which regions/continents are at the forefront of scientific progress in EEG-based BCI applications and highlight the most contributing authors on the topic.The objectives of the systematic literature review on EEG-based BCI applications:As per best practice, the PRISMA model ,31 has bThe publications concerning EEG-based BCI applications can be found from a high number of different sources. In the current review, three well-known databases, i.e., PubMed , Scopus During the full review of the 202 publications included in the analysis, 25 separate data items were extracted from each publication. The extraction of the data items follows the PRISMA model for preparing systematic literature reviews, and categorization of the extracted data items has also been applied in the past by Roy et al.  in a simSelection of the publications for the current review was based on pre-determined eligibility criteria. The eligibility criteria were determined to filter relevant publications concerning EEG-based BCI applications as per objectives of this review for further analysis. The eligibility criteria were selected according to similar principles used in previous review studies and studies that follow the PRISMA model ,28,29,30Publications needed to be relatively current. In the further analysis, publications were included from the period between 2009 and 2019.We excluded meeting abstracts, book chapters, masters and doctoral dissertations and non-English publications.We excluded non-peer-reviewed journal articles and non-peer-reviewed conference proceedings.The following eligibility criteria were applied during review of the publications during screening and eligibility assessment:Following the above-mentioned inclusion and exclusion steps, a manual scanning was conducted for the titles, keywords and abstracts of the publications. The screening process was conducted by one person. Publications that did not address the subject, but rather mentioned the subject in passing in general were excluded. During the review, 402 publications were excluded that did not meet the eligibility criteria. The scanning resulted in 233 publications for further full text assessment of eligibility. The overview concerning the process of identification, screening, assessing the eligibility and inclusion of the publications in the final analysis as per PRISMA model is shown in During the full text review of 233 publications, 31 publications which did not correspond to the eligibility criteria were further excluded. After all steps of publication selection per PRISMA model were completed, 202 publications were included  for the final analysis, which are ,215,216.The results have been divided into During the period from 2009 to 2019, the overall number of articles and conference proceedings published per year had been gradually rising. In the beginning of the period, the total number of the publications had been 11–15 publications per year from 2009 until 2011. During the period from 2012 to 2016, there was a slight increase in the volume of the publications to 16–19 publications, and only in year 2014 was there a decrease to 10 publications per year. Significant increases can be noted from year 2017 onwards when the number of publications per year had increased to up to 32 publications per year in 2017. Please see the overview concerning the number of articles and conference proceedings per year in The trend concerning increase in the overall number of publications per year has also been noted by Al-Nafjan et al.  when anaIt can be noted from The reason why the proportion of articles has increased over the recent years could be the overall development of the technology making it easier and more efficient to conduct the research on EEG-based BCI applications and inclusion of the topic more in the articles. In the beginning of the period under current focus, a higher variety and number of publications were included in conference proceedings. This trend noted in the current review illustrates the observation by Roy et al.  noting tThe distribution of the publications concerning EEG-based BCI applications were further analyzed by region/continent. Out of the 202 publications, the highest number of publications on the topic has been published in Asia (111 publications) where the highest number of publications per country has been published in China (39 publications).The other regions/continents where higher number of publications have been published on the topic during the period of 2009 to 2019 are Europe (56 publications) and North America (27 publications). In Europe, most productive countries have been Germany and Italy (both countries with 9 publications), and in North America, the highest number of publications have been published in the USA (23 publications). Similar results have also been found by Hwang et al.  and Roy Altogether, 37 different countries have contributed to the publications on EEG-based BCI applications within the time period reviewed. The highest number of contributing countries came from Europe, where the publications have been contributed by 18 different countries. From Asia, the publications have been contributed by 14 different countries, but the average number of publications per country is higher. From North America, publications have been contributed by three countries: USA, Canada and Mexico. In Australia, 7 publications were published, but from South America only one publication from Brazil has been published. It is interesting to note that no publications were contributed from Africa. It is important to note that the results reflect the publications matching the search terms used.Among the experimental publications on EEG-based BCI applications, non-medical publications have contributed the majority of the experimental publications per year. Exceptions to this trend have been seen in 2009 and 2013 when more medical publications were published when compared to the number of non-medical publications per year. Although in general there has been higher number of non-medical publications per year during the period under review, during recent years the proportion of medical publications has risen, reaching up to 36% of the publications.During recent years, the overall number of experimental publications has increased from 8–15 publications per year during 2014–2016 to 19–25 publications per year during the period of 2017–2019. The trend of a rapid increase in the overall number of publications on EEG-based BCI applications can also be seen in Hwang et al. .Throughout the period that was analyzed, there has been 99 publications (58% of the publications) published in the non-medical domain and 53 publications (31% of the publications) in the medical domain. In 19 cases (11% of the publications) the publications cover both domains. During interpretation of the results, it should be also noted that as the database search was conducted during the period from 20 October 2019 to 30 October 2019, the final number of publications for 2019 could have been slightly increased by the end of 2019. Please see the overview concerning the number of experimental publications per year in Although initially the EEG-based BCI applications were mainly developed for medical reasons to help patients to communicate, grasp objects, move around and support in other daily activities, the focus has been moving from the medical domain to non-medical applications. The shift in the focus does not reduce the importance of these applications in the medical domain but rather shows the wider potential of EEG-based BCI applications and opens doors for new possibilities.Among the publications within the period from 2009 to 2019, there were 58% of the publications (99 publications) from the non-medical domain. Among the overall number of publications that were analyzed, 31% of the publications (53 publications) are from the medical domain and in an additional 11% of the publications (19 publications) both domains have been included. Please see the distribution of the publications per domain in The publications in the medical domain could be further divided based on the type of applications into fields such as assistive, monitoring, rehabilitation, assessment and others. Applications in the assistive field help users with disorders or disabilities to perform daily tasks and provide assistance. Within the medical domain, the field covering assistive applications is the largest contributing 74% of the publications in the domain. The assistive field includes studies on robotic arm movement in a medical setting , lower-lThe publications concerning monitoring include for example monitoring emotional changes in patients . Among sThe prevalence of different fields in the medical domain differ when compared to previous work of Al-Nafjan et al. , as the The largest field in the non-medical domain is monitoring contributing 50% of the articles in the non-medical domain. Additionally, the two large fields are control machine and entertainment contributing 17% and 16% of the publications, respectively. Smaller coverage in the non-medical domain is by the publications in the field of assistive applications covering 4% of the publications and authentication covering 2% of the publications.Other different types of publications cover the rest of 11% of the publications in the domain. When compared to previous similar studies, Al-Nafjan et al.  has alsoIn the monitoring field, a total of 49 publications have been included. The publications in the monitoring field include studies where data are collected from the subjects in order to monitor and analyze their mental activity. The studies cover for example mental fatigue estimation , emotionIn a smaller number of the publications, both medical and non-medical domains have been covered. The domain has been further divided into the following fields: assistive, framework, control machine, monitoring and others, as per type of publications. The details concerning the distribution of publications covering both domains have been illustrated in Studies in the field of control machine are covering 16% of the studies. They include research for example on controlling a car in an experimental environment outside laboratory conditions . Some stAmong the studies analyzing EEG-based BCI applications, the most commonly used EEG devices are Emotiv EPOC from Emotiv , Quik-Cap from Compumedics Neuroscan  and MindWave from NeuroSky . These three devices combined cover 57% of the publications on the topic. The most common EEG device, the Emotiv EPOC , has been used in 40% of the publications analyzed while the prevalence of other popular EEG devices Quik-Cap  and MindWave  was 9% and 8%, respectively. The high prevalence of Emotiv EPOC  and Quik-Cap  use among studies analyzing EEG-based BCI applications for emotion recognition has also been noted by Al-Nafjan et al. .The most common EEG devices  are all manufactured in USA. Other EEG devices used in the studies have been manufactured also in Germany , Netherlands , Spain  and UK . The details of different EEG devices used, number of publications and percentage of publications covered is presented in Emotiv EPOC  and MindWave  are considered to be low-cost consumer EEG devices, while Quik-Cap  is more expensive to purchase . While tDadebayev et al.  have fouThe low-cost EEG headsets can be more convenient for the user and the devices such as Emotiv EPOC  and MindWave  have been successfully used in the studies developing EEG-based BCI applications . The finConcerning the design of the EEG devices it has been highlighted by Soufineyestani et al.  that theSimilarly, Hinrichs et al.  have comRatti et al.  have shoIn the study by Zerafa et al. , a compaWhen comparing the advantages and disadvantages of low-cost and more expensive EEG headsets LaRocco et al.  have fouAmong the studies analyzed, the number of EEG channels used varies across a large range. The highest number of EEG channels used was 163 by Zhou et al. for EEG-based classification of elbow versus shoulder torque intentions . AltogetPreviously, Roy et al.  found thAl-Nafjan et al.  have alsIn the publications, a variety of techniques are used for the interaction between brain and computer in BCI applications. The most popular techniques include the motor-imagery paradigm (applied in 38 publications), visual evoked potential paradigm (applied in 31 publications) and monitoring drowsiness/attention (applied in 29 publications) or emotions/affective states (applied in 15 publications). The overview concerning the prevalence of different techniques is shown in Among the publications applying motor-imagery paradigm, there were 62% of the publications in the medical domain and 24% of the publications in the non-medical domain. In addition to the aforementioned, 14% of the publications covered both the domains. Among the medical domain there are innovative applications such as EConHand , neuro-rIn the visual evoked potential paradigm, the non-medical domain is dominant as in 61% of the reviewed publications (where the technique used was stated) applied this technique. In the medical domain, the prevalence of this type of technique used was 23% and both domains were included in 16% of the cases. Visual evoked potential paradigm is widespread among BCI applications as it has been long used and tested in a high number of previous studies. The visual evoked potential paradigm also involves the visual P300 paradigm and the steady state visual evoked potential paradigm , use of There is significant interest in monitoring the mind for drowsiness/attention or emotions/affective states. Both techniques use the analysis of changes in the EEG spectrum to determine the states and changes in one’s mind. The vast majority of the publications concerning drowsiness/attention (88%) or emotions/affective states (93%) are categorized in the non-medical domain. The monitoring of drowsiness/attention based on EEG spectral changes has high practical value as there are a number of studies implementing the technique in order to create helmets for on-site workers for drowsiness detection , predictOther techniques used in the publications are applied less frequently, but there is a general trend noted in the overall use of various techniques. As in the case of motor-imagery, auditory evoked potential and vibrotactile evoked potential technique, the main application is in the medical domain, but the majority and larger variety of techniques are applied in the non-medical domain. The trend indicates the high potential of non-medical applications for the EEG-based BCI applications. When taking into consideration that the majority of general population apply for non-medical domain, vast number of people could benefit from this type of applications in the future.The feature extraction is an important processing step in order to extract the relevant details from the wide range of signals collected during acquisition of the EEG data. In previous studies, high number of different methods have been used for feature extraction. According to Al-Nafjan et al.  the featAmong the studies reviewed the most frequent feature extraction approaches are analysis of power spectral density (used in 23 publications), Fourier transform (used in 20 publications) or the analysis of common spatial pattern (used in 18 publications). Altogether, the aforementioned publications contribute to 48% of the reviewed publications. These results are similar to previous work by Al-Nafjan et al. , who staAccording to Xie et al.  the advaOther methods for feature extraction are used less frequently, but in many cases different feature extraction methods are used within one study. Similar general prevalence concerning the use of power spectral density and use of different feature extraction methods within studies has been noted by Al-Nafjan et al. , for anaOur overall results correlate with previous studies in the field highlighting the importance of analyzing power spectral density, the use of Fourier transform and common spatial pattern for feature extraction and emphasizing the need to apply different types of feature extraction methods depending on the application under study.For the purpose of classification, different machine learning algorithms have been used in previous studies for EEG-based BCI applications. The most common machine learning algorithms used are linear discriminant analysis and support vector machine, which have been applied in 52% of the studies. Other machine learning algorithms have the coverage of 2–5%. Among all of the methods, linear discriminant analysis has been applied in 31% of the cases and support vector machine in 21% of the studies. An overview of the classification methods employed in the reviewed studies is presented in Linear discriminant analysis has been characterized as a simple classifier with low computation requirements and acceptable accuracy . Linear Other methods applied in EEG-based BCI applications are machine learning algorithms such as convolutional neural network, naive Bayes and random forest that account for 4–5% of the cases and artificial neural network, deep neural network, Gaussian mixed model and k-nearest neighbors that account for 2–3% of the cases.The results of the current review are in correlation with previous studies where Al-Nafjan et al.  have shoDadabayev et al. , which pAccording to Nakagome et al. , when coIn the field of EEG-based BCI applications, there is a number of challenges hindering the development of the applications that would need to be addressed during future studies. It is important to acknowledge these aspects to find solutions or alternatives as needed. There are also many opportunities in the field to be used in the future and this is also an important aspect to highlight and share ideas among researchers. The sharing of new ideas and possibilities for the future facilitates the development of a large variety of applications in the field of EEG-based BCI applications.During the preparation of this systematic review, a strict process has been followed including the use of planned search terms and databases before the analysis of the literature. The time period for the publications was selected from 2009 until 2019 for conducting of the analysis in order to give an overview concerning a longer period of time. The review was designed in order to be able to compare the results with other previously conducted reviews on similar topics. Similar time interval has been selected also in previous studies ,28. The The selected search terms also set specific limitations for the publications identified and analyzed in the review. Using the term “EEG-based” could set specific limitations for the search results and term “EEG” could be used in order to obtain a wider variety of search results. The aforementioned use of specific search words could be the reason why some of the publications, for example ,232,233,The challenges for the BCI applications could be divided into technology-related and user-related, where technology-related challenges comprise technical aspects and the usability of the system and the user-related contain the aspects of a person to learn to use the BCI application and subjectiveness of the person to interpret the cues that generate or alter the acquired EEG signals . PadfielIn terms of challenges, the major aspect would be the current reliability of the BCI system in everyday noisy environments . This hiIn the medical domain (in the assistive field), the challenges include low recognition rate for mental commands , problemConcerning BCI applications in the non-medical domain, Wei et al. have highlighted that in order to transition laboratory-oriented BCI applications into real-world environments, the convenience to use and long-term wearing comfort for the devices needs to be improved . There hThe challenges hindering the development of BCI applications in both medical and non-medical settings involve poor portability of the hardware, low user-friendliness of the device, low signal quality and individual differences among persons that result in difficulties interpreting the EEG signals across users ,238,239.The use of current subject-dependent applications is time consuming due to training and especially inconvenient for people with mental disabilities . TherefoBoth medical and non-medical domains are affected by potential ethical issues in the EEG-based BCI applications filed of research. It would need to be determined for example who would be liable in case of any accidents during the use of BCI applications or could the applications affect, for example, user’s mood and therefore affect user’s decision making in a broader sense . It has It has been suggested that in order to make the EEG-based BCI applications more accurate and efficient hybrid BCI systems should be developed that combine BCI systems with another BCI or other kinds of interfaces . In addiIncreasing attention has been paid to fNIRS due to the advantages such as non-invasiveness, user safety, affordability and portability, but fNIRS signals are highly subject-specific and have low test-retest reliability . BarriosIn the medical domain, the BCI could be used to control a variety of assistive robotic devices. EEG-based BCI applications have been developed for controlling wheelchairs ,200, andIt has been further suggested to use virtual reality in neuro rehabilitation, where motor-imagery BCI virtual reality systems could be used for real time applications for stroke rehabilitation, to increase motivation of the patient during the rehabilitation process . The monIn the non-medical domain, there is a high potential for the EEG-based BCI applications monitoring cognitive load, attention, drowsiness and other aspects of the mind. Monitoring cognitive load has been studied by Friedman et al. for intelligence tests, but measuring cognitive load could also be used to better design and conduct e-learning, psychometric exams, military training and other trainings .Several studies have been published on monitoring attention via EEG-based BCI applications ,150,171.EEG-based BCI applications could be further developed for wider authentication of persons in addition to currently available methods . It has Entertainment is an ever-developing field and includes, for example, gaming. The availability and user friendliness of the games using EEG-based BCI has been increasing over time . With fuThe main reasons for the higher pace of development in the non-medical domain are due to the high potential of the domain for a wider range of users when compared to the medical domain. While development in medical domain is focusing on specific patient groups the EEG-based BCI applications in non-medical domain could be used by the general public, for example for smart home control, entertainment or gaming. The range of use for the devices in the non-medical domain is continuously broadening as new possibilities emerge for the use of the devices in new situations such as deceit identification test  or autheEEG-based BCI applications have a wide variety of applications in both medical and non-medical domains, with even higher potential recently in the non-medical domain due to a high number of potential users around the world. With the development of the technology, reduction in cost and increase in comfort of use the EEG-based BCI applications can gain increasing attention in the non-medical domain.During the daily use of EEG-based BCI applications, the safety of use is one of the most important aspects to focus on. In case of synchronous BCI, the controlling of the BCI is divided into time windows when the commands by the user could be given and the transfer of commands from the user to the device is well defined. For the user, asynchronous BCI applications would be preferred, as in case of asynchronous applications the commands could be given to the device at any time, independent of specific time windows. From the safety perspective, it is important to define, in case of asynchronous devices, when the user actually wanted to give command and when the user was thinking other thoughts not related to the use of the device. For this reason, the concept of “brain switch” would need to be further developed in practice, enabling the user to mentally disconnect from the device when the user is not intending to use the device ,131.Further possibilities for the EEG-based BCI applications include new, more convenient methods for obtaining EEG signals. Wei et al.  have sugIn the current review, both articles and conference proceedings have been included. The inclusion of both these types in the analysis gives better representation of the ideas in the field and helps reduce possible publication bias.Although initially the EEG-based BCI applications were mainly developed for medical purposes to help patients and support in daily activities, the focus has been moving from the medical domain to non-medical applications. The shift in focus does not reduce the importance of these applications in the medical domain but rather shows the wider potential of EEG-based BCI applications and opens new doors for applying the possibilities mode broadly.As per region, 111 publications (55%) have been contributed by Asia being firmly in the lead concerning number of studies published. From Asia most of the publications per country have been contributed by China. This is a relevant finding as China has increasing influence in general in high technology sector and EEG-based BCI could be one important field of research resulting in a high variety of high technology applications designed for many different fields of life, both medical and non-medical. The high potential of EEG-based BCI applications and diverse possibilities for use could support the future economic strength of the countries and regions investing in the research and development in the field.The reasons of the general popularity of Emotiv EPOC  EEG device are its relatively low cost, sufficient number of EEG channels and it being considered easy to use. Emotiv EPOC  and MindWave  are considered to be low-cost consumer EEG devices whereas Quik-Cap  is higher in cost. Emotiv EPOC  EEG device has 14 channels and MindWave  1 channel, whereas the Quik-Cap  EEG device uses 32 channels. MindWave  EEG device has limitations in use due to the limited number of EEG channels, but the low cost and ease of use make the device popular. The device could be applied in specific applications that do not require higher number of EEG channels. The Quik-Cap  device on the other hand is more expensive but has the advantage of higher number of EEG channels. The final decision concerning the use of a specific EEG device depends on the type of EEG-based BCI application, determining the need for specific number of EEG channels. The decision also depends on the cost planned for the study and the end users.When planning the selection of the EEG device and number of channels, it is important to consider the end users for the EEG-based BCI application, which determines the technical requirements and possible overall cost of the device. A smaller number of carefully selected electrode positions would also make the device more user friendly and support the performance of the system.There is a general trend noted in the overall use of various techniques. As in case of motor-imagery, auditory evoked potential and vibrotactile evoked potential technique the main application is in the medical domain, but the majority and larger variety of techniques are applied in the non-medical domain. As seen from the results of the current review, a highly diverse selection of techniques has been applied in the non-medical field supporting further the development of diverse applications and supporting the high potential of non-medical applications among the EEG-based BCI applications.In the current review, a trend of high prevalence of using power spectral density and Fourier transform for feature extraction has been noted. The overall results correlate with previous studies in the field, highlighting the importance of these methods for feature extraction and emphasizing the need to apply different types of feature extraction methods depending on the application under study. The results of the current review also show the importance of linear discriminant analysis and support vector machine for classification in correlation with previous studies in the field. The choice for classification algorithm depends on the type of brain signal being recorded and the type of application that is being controlled.With the widespread use of the devices, the safety on an individual and community level would need to be further analyzed. Nowadays, it has been unfortunately common that due to security breaches malicious software have infiltrated computer networks even with a high security level. This needs to be taken into consideration, especially when considering the sensitivity of the biomedical information obtained from the BCI device and also the possibility to alter the brain signals for example via neurofeedback. As the field of EEG-based BCI is fast developing, the ethical aspects would need to be analyzed and safeguarded in parallel with the development of technology.The BCI applications are developing rapidly and therefore it is important to have an up-to-date overview on the EEG-based BCI applications together with possible challenges and the way forward. The significance of the current review is to provide an overview of the current work conducted in the field of EEG-based BCI applications, along with the challenges and future possibilities.


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The shrinkage of foamed concrete increases with the increase in temperature; increasing the humidity of curing can control the water loss rate of foamed concrete and reduce shrinkage. Lower humidity and higher temperature will make cracks appear earlier; with an increase in the water–cement ratio, the initial cracking time is shortened and the cracking property of foamed concrete is improved. The industry standard “Technical Specification for Application of Foamed Concrete” (JGJ/T 341-2014) [Traditional foamed concrete is defined as a lightweight, porous concrete material, which is made by adding foam into slurry consisting of siliceous and calcareous components, water and admixture, followed by proper curing in certain conditions. The volume density of foamed concrete is 100–1600 kg/mIn recent years, China has continuously implemented reform of thermal insulation materials and building energy conservation policies, putting forward new requirements for thermal insulation materials and energy-saving materials, such as light weight, thermal insulation and high strength . Foamed 3/a, the design pressure is 12 MPa/10 MPa, and the pipe diameter is 1422 mm/1219 mm/101.6 mm. According to the overall direction of the route, the China–Russia East Route pipeline crosses the Yangtze River in Nantong Economic and Technological Development Zone and Changshu Economic and Technological Development Zone in Jiangsu Province, via the shield tunnel, which is the control project of the China–Russia East Route. The Yangtze River shield tunnel is huge, with a horizontal length of 10,324 m. The horizontal length of the shield tunnel crossing is 10226 m (north bank shaft center to south bank shaft center), as shown in The China–Russia Eastern trunk line starts from the China–Russia border in Heihe City, Heilongjiang Province, runs through Heilongjiang, Jilin, Inner Mongolia, Liaoning and Heilongjiang, Beijing, Tianjin, Shandong, Jiangsu, Shanghai, and ends at Baihe End Station in Shanghai, with a total length of 3334.6 km . The des2 isolation in foamed concrete brick, and the effect of bacterial self-healing and CO2 bio-sealing on the strength of bio foamed concrete brick [Domestic and foreign scholars have conducted much research on foamed concrete. Deng J. et al. studied the influence of different lengths and different dosages of polyvinyl alcohol fibers on the water absorption, strength and shrinkage of foamed concrete . Some scte brick ,18. Yangte brick . Hou Li te brick . Chen Y.te brick .Combined with research from scholars at home and abroad, it has been found that, in engineering applications, foamed concrete has many advantages when compared with traditional concrete; however, there is a lack of systematic research on the factors affecting the shrinkage and cracking of foamed concrete. In this paper, typical working conditions were simulated within different scenarios, and the shrinkage and maximum crack value of foamed concrete in these conditions were studied. This paper aims to systematically summarize the influence of curing system and water–cement ratio on the cracking and shrinkage of foamed concrete, and to provide theoretical guidance for the filling and construction of foamed concrete.At present, the problem of controlling the shrinkage cracking of foamed concrete is mostly seen in the research of additives, material ratio, fibers, etc. Based on this, this study formulates six curing systems, selects two water–cement ratios, and obtains the shrinkage and cracking performance of foamed concrete under different curing conditions and water–cement ratios, in order to systematically summarize the effects of temperature, humidity and water–cement ratio on the shrinkage and cracking of foamed concrete, and provide theoretical guidance for the filling and construction of foamed concrete.P.O 42.5 grade ordinary Portland cement was produced by Huaxin Cement  Co., Ltd., and the physical properties of the cement are shown in The fly ash, grade I, was sourced from Nanjing Huaneng Power Plant . The chemical composition of fly ash is shown in Foaming agent for HT foamed concrete is present in 3 foamed concrete are shown in Tap water is used, and, according to foamed concrete density grade A11, the compositions in 1 mHTFC foamed concrete admixture is provided by Henan Huatai New Material Technology Co., LTD. .The preparation of foamed concrete includes foam preparation and cement mortar preparation and casting . Foam waGiven the possible environments that foamed concrete may encounter in this project, six typical environmental conditions, including temperature and humidity, were selected to simulate the actual conditions of the project: (1) temperature 25 ± 1 °C and relative humidity 75 ± 5%; (2) temperature 25 ± 1 °C and relative humidity 15 ± 5%; (3) temperature 45 ± 1 °C and relative humidity 75 ± 5%; (4) temperature 45 ± 1 °C and relative humidity 15 ± 5%; (5) temperature 65 ± 1 °C and relative humidity 75 ± 5%; (6) temperature 65 ± 1 °C and relative humidity 15 ± 5%.(1)Steel molds measuring 40 × 40 × 160 mm were cleaned and lubricated for easier demolding. In addition, due to the high fluidity of foamed concrete, a layer of petroleum jelly was coated around each test mold to prevent the slurry from seeping out.(2)The foamed concrete slurry was cast in steel molds at the same temperature and humidity as stated in the designed curing regime.(3)The specimens were demolded after 24 h and placed in a curing box, with constant temperature and humidity control. The shrinkage of each specimen was measured after curing for different times, according to the test scheme. The test instrument and specimen are shown in (4)The drying shrinkage strain is determined as follows:The drying shrinkage of each specimen was measured 1 day after demolding, and continued for 28 days. The detailed test procedure was as follows:t, where t is the initial measurement time of drying shrinkage;t (mm).(1)The mixed foamed concrete was cast into an elliptical mold  and placed in a standard curing laboratory for curing.(2)The specimens were cured for 18 h in a curing room at 25 ± 1 °C with relative humidity 75 ± 5% before demolding.(3)When maintenance began, the strain gauge was pasted to collect data and observe the initial time of crack emergence. When the first crack was found, artificial observations were performed every 15 min for 12 h, then once a day afterwards.(4)The crack width of the elliptical ring of foamed concrete was observed with a 100-fold reading microscope, with a light source, and the development of the maximum crack width was observed and recorded over time.(5)The crack width was recorded with a reading microscope. The crack width was the average value of the crack width at 1/4, 1/2 and 3/4 of the height direction of the elliptical ring sample. The time of crack initiation, and the number and width of cracks in eadddch foamed concrete ellipse specimen were also observed and recorded. The test specimen is shown in The shrinkage data for foamed concrete during the curing regime were recorded using a comparator, as listed in The test data were analyzed by multi-factor analysis of variance, with mathematical statistical software SPSS  ; the resAccording to (1)When the humidity and water–cement ratio are the same, the 28-day shrinkage of foamed concrete increases with the increase in temperature;(2)When the humidity and temperature are the same, the 28-day shrinkage of foamed concrete decreases with the increase in water–cement ratio;(3)Temperature is the primary factor that affects the drying shrinkage performance of foamed concrete, followed by water–cement ratio and humidity.As can be seen from As the temperature rises, the shrinkage of the foamed concrete continues to increase. High temperature changes the drying shrinkage development process of foamed concrete. The drying shrinkage rate of foamed concrete increases rapidly in the early stage, and it then becomes stable afterward. In the initial stage, generally within the first 5 days, the shrinkage rate of foamed concrete is the largest, and with the extension of curing age, the shrinkage rate gradually slows down. When RH = 75%, the drying shrinkage of foamed concrete with different water–cement ratios tends to be the same, while when RH = 15%, the drying shrinkage is obviously different, due to different water–cement ratios. The shrinkage of foamed concrete obviously decreases with the decrease in water–cement ratio in a dry environment. At the same time, with the increase in water–cement ratio, the total shrinkage deformation of foamed concrete increases at 28 days.−6. When the temperature is 65 °C, the shrinkage difference of foamed concrete under different humidity is 66 × 10−6.As can be seen in As shown in As shown in Technical Regulations for Filling Lightweight Foamed Soil Rail Transit” (CECS453-2016) [In the case study of the China–Russian Eastern Gas Pipeline, the shrinkage and cracking of foamed concrete was studied under typical environments, where temperature, humidity and water–cement ratio were representative of project scenarios. The results show that the decrease in humidity and the increase in temperature will reduce the shrinkage and cracking of foamed concrete. During the test, it was found that the range of water–cement ratio 0.4–0.5 can meet the shrinkage and cracking requirements of foamed concrete. In simulated conditions, the maximum crack width of foamed concrete was 3.3 mm, which meets the requirements of the “53-2016) . The wid53-2016)  that nee53-2016)  and prop


Antibiotic use at the end of life is common in adult patients but little is known about children. While important to treat and prevent infection in these vulnerable populations, unnecessary antibiotic exposure may result in adverse events and selection of antimicrobial resistant organisms. The purpose of this study was to describe clinical features of pediatric patients who receive antibiotic use at the end of life, quantify antibiotic utilization, and determine indications for antibiotic use.We performed a retrospective review of antibiotic use in the last 30 days of life who had palliative care consultation at a tertiary care pediatric hospital from June 2012 to February 2020. Demographic characteristics, primary diagnosis, do not resuscitate (DNR) status, and hospice status were assessed. Days of antibiotic therapy were recorded.Of the 151 patients identified, 67 (44%) were female, and the median age at death was 3.5 years (IQR: 0.50-13.8 years). Of the causes of death, 133 (88%) had chronic progressive illness, 13 (8.7%) had a chronic static illness, 5 (3.3%) and had an acute illness. The most common primary diagnoses were oncologic , cardiac , genetic metabolic , and neurological . DNR status was recorded for 109 (72%) patients, and 11 (7.2%) had documented hospice status. Antimicrobial use was most common for a known organism , followed by no documented indication , empiric therapy , and prophylaxis only (n=2.7%). Of the 2126 total days of therapy of antibiotic use, the most common agents were vancomycin (n=379), meropenem (n=254), ceftazidime (n=171), and cefepime (n=143). The median number of unique antibiotics received in the last 30 days of life was 2 (IQR 0-5).Antibiotic exposure was common in pediatric patients at the end of life, particularly for vancomycin and meropenem, even without documented infection. Opportunities for future interventions include better discussion of goals of care between providers and parents and risk of adverse events, particularly if suspicion of infection is low.All Authors: No reported disclosures.


Based on an examination of its antimicrobial activity, the fn-POSS blended TS (TSP-5.0) film exhibits a favorable zone of inhibition against the bacterial pathogenic Staphylococcus aureus and Escherichia coli. The TS/fn-POSS (TSP-5.0) film lost 78.4% of its weight after 28 days in natural soil. New plastic materials used for packaging, especially food packaging, are typically not biodegradable, so the TS composite with 5.0 wt.% fn-POSS is therefore of definite interest. The incorporation of fn-POSS with TS composites can improve their characteristics, boost the use of nanoparticles in food packaging, and promote studies on biodegradable composites.Rapid advancements in materials that offer the appropriate mechanical strength, barrier, and antimicrobial activity for food packaging are still confronted with significant challenges. In this study, a modest, environmentally friendly method was used to synthesize functionalized octakis(3-chloropropyl)octasilsesquioxane [fn-POSS] nanofiller. Composite films compared to the neat thermoplastic starch (TS) film, show improved thermal and mechanical properties. Tensile strength results improved from 7.8 MPa to 28.1 MPa (TS + 5.0 wt.% fn-POSS) with fn-POSS loading (neat TS). The barrier characteristics of TS/fn-POSS composites were increased by fn-POSS by offering penetrant molecules with a twisting pathway. Also, the rates of O Soft materials based on bioplastics have been used for a long time to produce food containers, containers for storing food and packing for food products ,2,3,4,5.Two carbohydrates combine to make a semicrystalline material called starch, with several branches, and amylose, which is a linear molecule . The amoTo meet the demands for food packaging, factors such as low cost, processability, and the amount of raw material required have all been studied in the case of hydrocarbon plastics ,20,21,22To increase the mechanical and physical intestinal barrier and antimicrobial activity, biopolymers have been combined with nanoparticles (NPs), such as inorganic metal and metal oxides, CNTs, graphene oxide, and montmorillonite ,30,31. FIn this work, solution casting has been used to produce TS/fn-POSS composite films, with fn-POSS synthesized under microwave-assisted conditions. To study the structure and morphologies of TS/fn-POSS composites, instruments including scanning electron microscopy (SEM) and Fourier transform infrared (FTIR) spectroscopies have been used. However, morphologies and physicochemical properties, such as mechanical, oxygen transmission rate (OTR), and water vapor transmission rate (WVTR), as well as antimicrobial activity, are strongly affected by fn-POSS contents. The clean starch film and composites’ morphological and chemical characteristics, as well as mechanical and thermal stability, have also been determined. The results indicated that TS/fn-POSS composites can be used more significantly in the food packaging sector with decreased barrier properties and increased mechanical, thermal, and antimicrobial activity.−1 and 970 cm−1, respectively. By bending CH2, the FTIR peak at 1487 cm−1 is formed. There is confirmation of the Si-O-Si, C-O vibration in the peaks at 795 and 650 cm−1, accordingly. The fn-POSS revealed a spectrum at 3000 cm−1 which was linked to the -OH groups’ distortion, perhaps as a result of the absorbed water molecules. The (101), (004), and (200) crystal planes are indicated by the diffraction peaks at 25.3°, 38.3°, and 48.0°. 2 and H2O.FTIR spectroscopy was used to confirm the synthesis of the fn-POSS, as shown in −1, which was formed by bound C-H that was loose on the starch which is stretched, both inter- and intramolecularly. The stretching of the -CO bond led to peaks in the starch wavelength at 1183, 1090, and 922 cm−1, respectively. A suggestion for the peak at 1040 cm−1 is the Si-O-Si stretching vibration. The 1740 cm−1 peak relates to the stretching vibrations of C=O. The unique absorption peaks which are connected to the TS polymer units can be observed in the spectra of the TS/fn-POSS composite films. In other words, the FTIR spectra of the TS/fn-POSS composites indicate that after the combination of fn-POSS, the film remains, clearly showing the distinct and unique peaks of TS. This is provided that the FTIR bands of the TS and TS/fn-POSS composites were alike, or that there is no interaction between TS and fn-POSS or the POSS typical peak and the TS characteristic peak overlap. The structural characteristics of the composites composed of TS and TS/fn-POSS were tested using XRD. The XRD patterns of TS fn-POSS nanostructures with various weight % are represented in The FTIR of the TS and TS/fn-POSS composite materials are shown in The results of the SEM analysis are shown in 2 and H2O barrier properties, in addition to its mechanical performance, are a significant indication for food packaging materials [2/day. With an increase in fn-POSS in the TS matrix, the WVTR values decreased. The lowest WVTR value for TSP-5.0 was 48.1 g/m2/day because oxygen molecules find it difficult to permeate the TS matrix when fn-POSS NPs are present; one of the most essential components of packaging material is its oxygen permeability. The results of measuring the OTR values of TS/fn-POSS nanocomposites are shown in 2/24 h. The films’ barrier characteristics were enhanced by adding fn-POSS to the TS matrix. The interaction of two phenomena produced diffusion through the polymers: The area allowed for gas diffusion decreased as fn-POSS NPs were loaded, and the distance that gaseous molecules must travel from across films increased. It was found that the OTR values of the nanocomposites were lower than the TS matrix. The addition of the fn-POSS did not specifically improve the crystallinity of the TS in this investigation. The higher tortuosity of the oxygen molecule’s diffusion path, given by the evenly distributed fn-POSS, is thought to be the reason for the nanocomposites’ lower OTR . When fn-POSS is added, the OTR value of the nanocomposite decreased by up to 5.0 wt.%. Similar reports have been published about the other nanoparticle/polymer systems as well [The mechanical strength of the TS/fn-POSS composite is dependent on fn-POSS NPs, as seen in aterials . Figure  as well , where oTo study the surface characteristics of TS/fn-POSS composites and identify the effects of the filler content, wettability measurements, or WCA, were performed. S. aureus and E. coli were used as a test group for the antimicrobial activity of TS and TS/fn-POSS composites. The results are represented in S. aureus and E. coli. The TS/fn-POSS composites, in contrast, show an obvious antimicrobial zone of inhibition. The diameters of the antimicrobial zones in the TS/fn-POSS composites are depicted in S. aureus and E. coli suggests that the addition of fn-POSS can improve antimicrobial activity. Additional confirmation that the TS/fn-POSS composite film effects are real and significant against S. aureus microorganisms is provided by the somewhat smaller antimicrobial zone widths for S. aureus greater in comparison to those for E. coli and S. aureus. This was tested to confirm the zone of inhibition activity of TS in the TS/fn-POSS composite, and the results were similar to all those stated in other reports [Over recent years, composites based on nanoparticles have become increasingly used in biological applications for their antimicrobial activity ,44. The  reports . Accordi reports ,47, the  reports ,49.Biodegradable packaging films fabricated from renewable materials are required to preserve food quality, be long-lasting, and be less wasteful in their manufacture . ChangesThe thermoplastic starch in this study was obtained from S.D. Fine Chemicals in India. The majority (97%) of the obtained 3-chloropropyl trimethoxysilane was obtained from Alfa Aesar in India. Acetic acid, dichloromethane, and methanol (99%) were purchased from Sigma Aldrich in India. All chemicals obtained were used without any further purification.−1) (C-C stretching), 1110 (cm−1) (Si-O-Si stretching vibrations), 970 (cm−1) (C-O stretching vibrations), 795, and 650 (cm−1) (bending vibrations of Si-O-Si). The characterization information agrees with the previous article [As per earlier research , the cha article .The TS/fn-POSS composites were produced by solution mixing and drop-casting, as shown in −1 (Perkin Elmer spectrophotometer RX1). The 2 × 2 cm squares of the prepared specimens were instantly sliced and placed in the sample holder. The wavenumber band for the spectra was 4000–400 cm−1.An FTIR spectrophotometer was used to determine the film sample’s FTIR spectrum at a resolution of 4 cmThe composites were tested by XRD at room temperature using an analytical diffraction meter . After the specimens were prepared according to the instructions and deposited (2 × 2 cm) on a glass plate, the spectra were collected.With the help of a microscope with a 5.0 kV accelerating voltage, the TS/fn-POSS and fn-POSS NPs were micrographed by SEM. The TS/fn-POSS composite samples were distributed on a carbon tape surface. Prior to imaging, a gold sputter coating was applied to every sample. The TEM test was carried out using an electron microscope with a 200 kV acceleration voltage (FEI TECNAI T-20).To evaluate the thermal stability, a thermogravimetric tester  was used. All samples were heated at a rate of 10 °C/min to temperatures ranging from 25 to 700 °C in a nitrogen atmosphere.A universal testing machine  was used to test the tensile strength of materials with the following aspects: 25 mm wide, 50 mm in length, and 0.085 mm thick (ASTM-D882-02). After the examination of five samples, the average value was calculated.An oxygen permeability tester was used to evaluate the OTR of the TS/fn-POSS composites at 25 °C . The composites were measured several times in various locations to get at the average value. All tests were performed in daylight.The WVTRs of the fn-POSS nanoparticles combined with TS were investigated and measured using a water vapor permeability analyzer . All water vapor permeability tests were performed at room temperature in accordance with the ASTM F-1249 standards.The water contact angle of the TS/fn-POSS composites was evaluated using a goniometer  and the sessile drop method at 23 °C and 50% RH. To determine the contact angle, a 1 L droplet of water was dropped onto the coated surface, and an image of the droplet was recorded within 5 s. The mean value was calculated as the average of the contact angle measurements made at five different locations on the film. The experiment’s error rate was 1°.E. coli and S. aureus, were used to assess the antimicrobial activities of the films in their as-prepared state. Professor Jintae Lee of Yeungnam University in the Republic of Korea donated the films on the E. coli and S. aureus strains  for in vitro studies. The films were originally purchased from the American Type Culture Collection (ATCC). The culture media, microbial inoculum size, and incubation conditions were in accordance with the CLSI’s recommendations [The method of disk diffusion was used to investigate the antimicrobial activity of the TS matrix and its composites ,60. Two ndations . After to and Wt represent the pre- and post-burial weights of the specimens in the compost soil. According to the average results from up to five specimens for each of the fn-POSS contents, the final results were reported.The evaluation of biodegradation was performed using a method that has been described in the literature ,63,64. TTo show repeatability, all studies were conducted five times. The mean and standard deviation of all the statistical data were calculated using SPSS 22.0. To determine the major differences between groups, a one-way analysis of variance (ANOVA) was used. For statistical significance, a difference had to be below 0.05. SigmaPlot was used to present the results.S. aureus and E. coli). Therefore, the TSP-5.0 film may be proposed as a suitable organic fruit and vegetable packing film. The results indicate that TS/fn-POSS composites can be utilized as materials for food packaging which minimize the microbiological load and extend the shelf life of packaged foods.In the current study, solution casting was employed to produce TS/fn-POSS composite films. The FTIR results showed that there was intermolecular hydrogen bonding between the TS matrix and fn-POSS. The uniform and homogeneous dispersion of fn-POSS on the TS matrix in TEM and SEM micrographs was confirmed by TSP-3.0. The elongation at break and tensile strength of the TSP-5.0 film increases due to the addition of fn-POSS to the TS matrix, raising its mechanical strength and allowing it to be studied as a structurally stable food packaging film. The WVTR and OTR for TS/fn-POSS composites were substantially lower compared to TS. By enhancing the water contact angle, fn-POSS also produced TS materials which were more hydrophobic. By increasing the amount of fn-POSS, the TS’s water contact angle improved from 56.4° to 76.3° which enhanced hydrophobicity. The TS/fn-POSS composite film displayed an impressive antimicrobial action against microorganisms, which increased with increasing fn-POSS (


Triplophysadaryoae, new species, is described from the Sokh River, a former tributary of Syr Darya that today fails to reach the river, in the Sokh District, an exclave of Uzbekistan, surrounded by Kyrgyzstan. Triplophysadaryoae is distinguished from other species of Triplophysa in Central Asia by a truncate caudal fin with 13 or 14 branched rays, body without obvious mottling, dorsal-fin origin opposite to pelvic-fin insertion, and absence of the posterior chamber of the air bladder. Molecular data suggest that Triplophysadaryoae is closely related to T.ferganaensis from the Shakhimardan stream, a small tributary of Syr Darya in the Yordon village, another exclave of Uzbekistan in Kyrgyzstan. The two species were separated by a Kimura 2-parameter genetic distance of 2.8% in the mitochondrial DNA cytochrome c oxidase subunit I barcode region; they are also distinguished morphologically. A key to the species of Triplophysa in the Syr Darya basin and adjacent regions is provided. Triplophysa Rendahl, 1933, comprises approximately 160 species , T.elegans , T.ferganaensis Sheraliev & Peng, 2021, and T.strauchii  have been recorded from the rivers of the Fergana Valley  in the Fergana Valley is controversial and requires additional in-depth taxonomic research , guidelines, and policies approved by the Southwest University Local Ethics Committee for Animal Experiments. After euthanasia, specimens were fixed in 10% formalin and stored in 70% ethanol. The right-side pectoral fin was preserved in 95% ethanol for molecular analysis. Counts and measurements were performed following the procedures of SWU) in Beibei, Chongqing, China, and the private Bakhtiyor Sheraliev Fish Collection (BSFC) in Fergana, Uzbekistan.Handling of specimens was consistent with the Republic of Uzbekistan Animal Welfare Laws , T.ulacholica , and T.coniptera  were obtained from T.sewerzowi  from T.dorsonotata  and T.lacusnigri  from T.kungessana  from Triplophysa species from the Tarim basin, data on T.laterimaculata Li, Liu & Yang, 2007, T.papillosolabiata , T.bombifrons , T.zamegacephala , T.moquensis Ding, 1994, T.orientalis , T.microphysa , and T.incipiens  were obtained from T.herzensteini , T.microphthalma , T.kaznakowi Prokofiev, 2004, and T.waisihani Cao & Zhang, 2008 were obtained from BSFC, FSU, ICIZ, NWIPB, and SWU.Data on CPD, caudal-peduncle depth; CPL, caudal-peduncle length; HL, head length; K2P, Kimura 2-parameter; SL, standard length.Abbreviations: BSFC, Bakhtiyor Sheraliev Fish Collection, Fergana, Uzbekistan;FSU, Fergana State University, Faculty of Life Sciences, Fergana, Uzbekistan;ICIZ, Ichthyological Collection of the Institute of Zoology, Academy of Sciences of Uzbekistan, Tashkent, Uzbekistan;NWIPB, Northwest Institute of Plateau Biology, Chinese Academy of Sciences, Qinghai, China;SWU, Southwest University, School of Life Sciences, Chongqing, China.Collection codes:c oxidase subunit 1 (COI) barcode region (652 bp) was amplified using primers VF2_t1 (5'-TGT AAA ACG GCC AGT CAA CCA ACC ACA AAG ACA TTG GCA C-3') and FR1d_t1 (5'-CAG GAA ACA GCT ATG ACA CCT CAG GGT GTC CGA ARA AYC ARA A-3'), as designed by DNA was extracted from the right-side pectoral fin using proteinase K digestion followed by a standard phenol-chloroform method . The mitCOI gene sequence was used for phylogenetic analysis. Molecular analysis was conducted using three new COI sequences , OK377302), as well as 29 previously published sequences retrieved from the National Center for Biotechnology Information (NCBI) GenBank (Table https://www.ncbi.nlm.nih.gov). The COI sequences were aligned using the Clustal_W algorithm in MEGA7  using MrBayes ver.3.2 (ML) using MEGA7. MrBayes was run with six substitution types (nst = 6), considering a general time-reversible model with gamma-distributed rate variation and proportion of invariable sites (GTR+G+I) for the COI datasets. For BI analysis, we ran four simultaneous Monte Carlo Markov chains for 3,000,000 generations, with sampling every 1000 generations. The chain temperature was set as 0.2. Log-likelihood stability was determined after 10,000 generations, and the first 1000 trees were excluded as burn-in. The remaining trees were used to compute a 50% majority-rule consensus tree. For ML analysis, we conducted heuristic searches (1000 runs) also using the GTR+G+I model (nst = 6). Phylogenetic trees were visualized and edited using FigTree ver.1.4.2 , (MF172073), and B.toni  (KX039660) were used as outgroup.The 652 bp in MEGA7 , with ma ver.3.2  and maxier.1.4.2 . BarbatuTaxon classificationAnimaliaCypriniformesNemacheilidaeE79D7931-3079-5A87-9FFE-2BED7C63B180http://zoobank.org/8CE5BCB5-F671-4270-BFA3-7884DEF0BED7SWU 20211207001, male, 78.5 mm SL; Uzbekistan, Fergana Region, Sokh District, Sokh River, near Limbur village, an exclave of Uzbekistan surrounded by Kyrgyzstan, Syr Darya basin, 40°3.1528'N, 71°5.8195'E, altitude 1054 m, December 07, 2021, collected by B. Sheraliev and Y. Kayumova.SWU 20211207002-011, 10, 49.0–94.0 mm SL; BSFC 0023, 4, 62.1–82.4 mm SL; Uzbekistan, Fergana Region, Sokh District, Sokh River, near the Limbur village, exclave of Uzbekistan, Syr Darya basin, 40°2.7387'N, 71°6.288'E, altitude 1054 m, April 12, 2021, collected by Y. Kayumova. BSFC 0024, 3, 74.1–81.3 mm SL, same data as holotype.Triplophysadaryoae is distinguished from congeners by a combination of characters. It is distinguished from T.ferganaensis by possessing a truncate caudal fin with 13–14 branched rays , 9 pores in the pre-opercular mandibula (vs 7–8), and a slenderer body . It is distinguished from T.strauchii by absence of the posterior chamber of the air bladder , possessing 9–10 inner gill rakers on the first gill arch (vs 12–16), and no obvious skin mottling (vs mottling). Triplophysadaryoae is also distinguished from T.dorsalis, T.dorsonotata, and T.elegans by having a truncate caudal fin (vs emarginate) and lacking a posterior chamber of the air bladder . It is distinguished from T.sewerzowi, T.tenuis, and T.ulacholica by the dorsal-fin origin opposite to the pelvic-fin insertion .T.daryoae are given in Table N = 2); gill rakers, 9–10 in the inner row of first gill arch (N = 4). Cephalic lateral-line system, 2 supratemporal, 6 supraorbital, 4+10–11 infraorbital, and 9 pre-operculum mandibular pores.Morphometric data of SL. Head compression, maximum width always greater than depth; head maximum width 63.2–73.2% of HL. Snout slightly pointed, length shorter, equal, or slightly longer than postorbital length; snout length 34.9–47.3% of HL. Anterior and posterior nostrils adjacent; anterior nostril as short tube with elongated barbel-like tip; tip of nostril barbel not reaching the anterior margin of eyes. Eyes normal; diameter 12.5–17.1% of HL , T.dorsalis, T.dorsonotata, T.elegans, T.ferganaensis, T.sewerzowi, T.strauchii, T.tenuis , and T.ulacholica. The minimum K2P distances between T.daryoae and its closest relatives T.ferganaensis and T.tenuis were 2.8% and 4.5%, respectively   to the previously published molecular reconstructions , 6 branched pelvic-fin rays (vs 7 or 8), 9–11  branched pectoral-fin rays ; cephalic lateral-line system with 6 suborbital and 9 pre-operculo mandibular pores , dorsal and caudal fins almost hyaline, and spots imperceptible . Triplophysadaryoae is distinguished from T.strauchii, which is the most common species of Triplophysa in the waters of Fergana Valley, by the small number of vertebrae (4+35 vs 4+37–38); a smaller number of gill rakers in the inner row of the first gill arch (9 or 10 vs 12–16); absent posterior chamber of air bladder ; shorter body depth and width at dorsal-fin origin ; dorsal-fin origin equal to pelvic-fin insertion ; and upper and lower lobes of caudal fin equal (vs upper lobe slightly longer than lower lobe). Triplophysadaryoae differs from T.dorsalis and T.elegans, which are rare species in the Fergana Valley, according to the following characteristics: dorsal-fin origin equal to pelvic-fin insertion ; 9 or 10 gill rakers in the inner row of the first gill arch ; wider interorbital width ; longer pectoral-pelvic distance ; shorter dorsal-fin depth ; caudal peduncle equal to HL ; caudal-peduncle depth 7.6–9.2% of SL (vs 6.6–7.1% of SL in T.elegans); posterior chamber of air bladder degenerated (vs developed in both); lips thick with furrows ; and caudal-peduncle depth 2.2–2.9 times its length (vs 3.2–3.5 times in T.elegans). Moreover, the genetic distance between the TriplophysadaryoaeT.dorsalis, T.elegans, T.ferganaensis, and T.strauchii , based on the mitochondrial COI barcoding region, is consistent with the species-level divergence in other fish taxa ; dorsal fin slightly posterior to the midpoint between the tip of the snout and caudal-fin origin ; caudal-peduncle depth 7.6–9.2% of SL (vs 5.2–5.7% of SL), head depth 52.1–60.6% of HL (vs 47.8–51.2% of HL); and caudal-peduncle depth 2.2–2.9 times its length (vs 4.1–4.3 times). Triplophysadaryoae is distinguished from T.paradoxa by the absence of scales , dorsal-fin origin opposite to pelvic-fin insertion , and dorsal-fin origin closer to the caudal-fin base than to the snout tip (vs closer to the snout tip). Triplophysadaryoae is distinguished from T.ulacholica by the shorter average caudal-peduncle length (20.8 ± 1.11% of SL vs 27.0 ± 0.24% of SL), deeper caudal peduncle (7.6–9.2% of SL vs 4.9–6.8% of SL), caudal-peduncle depth 2.2–2.9 times its length (vs 4.0–5.3 times), and smaller eye diameter (12.5–17.1% of HL vs 18.5–23.3% of HL). The new species can be distinguished from T.coniptera by the rounded edge of the pectoral fin (vs pointed); caudal fin truncate (vs deeply forked); snout length 7.4–10.0% of SL (vs 10.1–11.8% of SL); caudal-peduncle depth 7.6–9.2% of SL (vs 5.2–7.3% of SL); and caudal-peduncle depth 2.2–2.9 times its length (vs 3.4–4.5 times). Triplophysadaryoae is distinguished from T.sewerzowi by the following characteristics: dorsal-fin origin opposite to pelvic-fin insertion , second branched ray of dorsal fin the longest (vs third or fourth), and degenerated posterior chamber of the air bladder (vs developed). It is also distinguished from T.dorsonotata by its smaller eye diameter (7.2 times of HL vs approximately 6 times), pre-dorsal length longer than post-dorsal length , cephalic lateral-line system with 6 suborbital pores (vs 7 or 8), and caudal fin truncate (vs emarginated). The new species can be distinguished from T.kungessana by its thick lips with furrows and papillae  and pelvic fin reaching the anus (vs not reaching the anus). Triplophysadaryoae can be distinguished from T.herzensteini by having 4+35 vertebrae (vs 4+39–41) and a caudal-peduncle depth 7.6–9.2% of SL (vs 5.9–6.8% of SL). Triplophysadaryoae can be distinguished from T.uranoscopus by its caudal-peduncle depth measuring 2.2–2.9 times its length (vs. 2.7–4.1 times); caudal-peduncle depth 7.6–9.2% of SL (vs 5.4–7.0% of SL); dorsal-fin origin opposite to pelvic-fin insertion . It differs from T.lacusnigri by a shorter head length (4.3–5.0 times SL vs 3.4–4.5 times SL) and non-oblique head profile in front of the eye (vs strongly oblique); the tip of the pectoral fin is usually formed by 4 branched rays (vs 2 or 3).Another ten species of a Valley . Among tTriplophysadorsonotata, T.elegans, T.lacusnigri, T.tenuis, and T.uranoscopus from different water bodies in Central Asia have long been synonymized with T.stolickai due to their morphological resemblance  from T.daryoae of 6.3%, 6.8%, and 9.7% ; interorbital width being 29.5–35.6% of HL (vs 20.8–26.2% of HL); head depth at nape 52.1–60.6% of HL (vs 46.1–51.5% of HL in Ili River); caudal-peduncle depth 2.2–2.9 times its length ; vertebrae 4+35 (vs 4+38–41); body without obvious mottling (vs. with mottling); supraorbital sensory canal always connected with the infraorbital canal ; and 2 supratemporal pores (vs 3 or 4).emblance . We werebekistan . To confTriplophysadaryoae is similar to some species of Triplophysa from the Tarim basin and adjacent regions, with normal eyes, scaleless body, body color, color patterns, and slightly laterally compressed caudal peduncle ; 9 or 10 gill rakers in the inner row of the first gill arch ; dorsal fin inserted opposite to vertical through pelvic-fin origin ; pelvic fin reaching the anus (vs not reaching in T.kaznakowi); pre-pelvic length 50.7–55.5% of SL (vs 56.3–60.5% of SL in T.laterimaculata); caudal-peduncle length 19.1–23.1% of SL (vs. 28.6–31.3% of SL in T.bombifrons and 23.3–29.4% of SL in T.papillosolabiata); caudal-peduncle depth 7.6–9.2% of SL (vs. 4.5–6.7% of SL in T.waisihani); and 4+35 vertebrae (vs 4+38–43 in T.moquensis and 4+39–41 in T.waisihani). T.daryoae can be distinguished from other Triplophysa species by the following characteristics: 39 vertebrae ; caudal-peduncle depth 2.2–2.9 times its length ; and posterior chamber of air bladder degenerate .peduncle . HoweverTriplophysadorsalis species group is proposed here based on the molecular data and phylogenetic reconstruction obtained. The proposed species (see above) have also been nested in a single clade in previous phylogenetic studies of Triplophysa , T.dorsonotata (Ili River), T.herzensteini (Ili River basin), T.kungessana (Künes River), T.paradoxa  and T.salari (Chirchik River) also belong to this species group. In contrast, T.uranoscopus, which is widely distributed in the Zeravshan River, was not nested in this clade  in the Amu Darya basin may serve as an important key to understanding how Triplophysa species have evolved in Central Asia.A plophysa . Howeverade Fig. . This reT.dorsalis: FSU uncatalogued, 6, 56.2–83.5 mm SL; Kara Darya, Andijan Region, Uzbekistan; ICIZ 2200016, 11, 51.6–88.8 mm SL; Achangaran River, Tashkent Region, Uzbekistan.T.elegans: SWU 20190818630–634, 5, 47.9–69.1 mm SL; Chirchik River, Tashkent Region, Uzbekistan.T.ferganaensis: SWU 20190813001, holotype, 87.5 mm SL; Shakhimardan stream in Yordon village, Syr Darya basin, Fergana District, Fergana Region, Uzbekistan. SWU 20190813002–021, FSU 082019650–654, 25 paratypes, 49.5–109.2 mm SL; Shakhimardan stream in Yordon village, Syr Darya basin, Fergana District, Fergana Region, Uzbekistan. BSFC 0025, 2, 42.9–66.2 mm SL; Shakhimardan stream in Yordon village, Syr Darya basin, Fergana District, Fergana Region, Uzbekistan.T.stolickai: NWIPB 1305044, 1305046–48, 1305052, 1305056, 1305060, 7, 56.0–102.5 mm SL; Kashi River, Nilka County, Ili River System, Xinjiang Province, China. NWIPB 1305111, 1305113–115, 4, 67.6–91.0 mm SL; Künes River, Xinyuan County, Ili River System, Xinjiang Province, China. NWIPB 1305131, 1305141–142, 3, 64.0–75.4 mm SL; Tekes River, Tekes County, Ili River System, Xinjiang Province, China. NWIPB 1307006–007, 1307014, 3, 58.3–79.5 mm SL; Zhaqu River, Chindu County, Yangtze River System, Qinghai Province, China. NWIPB 1407013–018, 6, 62.7–84.2 mm SL; Changchuan River, Rutog County, Indus River System, Tibet Autonomous Region, China. NWIPB 1007083, 1, 97.5 mm SL; Yarkand River, Yecheng County, Tarim River System, Xinjiang Province, China. NWIPB 1007084, 1, 73.9 mm SL; Qaraqash River, Pishan County, Tarim River System, Xinjiang Province, China.T.strauchii: SWU 20190820642–644, 3, 74.0–110.5 mm SL; unnamed stream, Syr Darya River System, Fergana District, Fergana Region, Uzbekistan. SWU 20190809551, 1, 69.5 mm SL; Kara Darya, Andijan Region, Uzbekistan. SWU 20190818617–642, 26, 45.9–98.7 mm SL; Chirchik River, Tashkent Region, Uzbekistan. BSFC 0022, 8, 83.6–155.9 mm SL; Great Fergana Canal, Syr Darya River System, Uzbekistan District, Fergana Region, Uzbekistan.T.tenuis: NWIPB1250170–174, 5, 83.2–111.2 mm SL; Heihe River, Zhangye city, Heihe River System, Gansu Province, China.T.uranoscopus: SWU 20190802503–504, 2, 78.0–80.2 mm SL; Zeravshan River, Samarkand Region, Uzbekistan; BSFC 0041, 8, 51.5–96.1 mm SL; Karadarya River, Zeravshan River System, Oqdaryo District, Samarkand Region, Uzbekistan.


MET) exon 14 skipping for the clinical stratification of non-small cell lung cancer (NSCLC) patients. Different technical approaches are available to detect MET exon 14 skipping in routine practice. Here, the technical performance and reproducibility of testing strategies for MET exon 14 skipping carried out in various centers were evaluated. In this retrospective study, each institution received a set (n = 10) of a customized artificial formalin-fixed paraffin-embedded (FFPE) cell line (Custom METex14 skipping FFPE block) that harbored the MET exon 14 skipping mutation , which was previously validated by the Predictive Molecular Pathology Laboratory at the University of Naples Federico II. Each participating institution managed the reference slides according to their internal routine workflow. MET exon 14 skipping was successfully detected by all participating institutions. Molecular analysis highlighted a median Cq cut off of 29.3 (ranging from 27.1 to 30.7) and 2514 (ranging from 160 to 7526) read counts for real-time polymerase chain reaction (RT-PCR) and NGS-based analyses, respectively. Artificial reference slides were a valid tool to harmonize technical workflows in the evaluation of MET exon 14 skipping molecular alterations in routine practice.Lung cancer remains the leading cause of cancer deaths worldwide. International societies have promoted the molecular analysis of MET proto-oncogene, receptor tyrosine kinase ( Lung cancer still represents the leading cause of cancer deaths worldwide . RemarkaAmong the different genomic alterations, including point mutations, insertions/deletions (indels), and gene fusions, a novel class of molecular aberrations should be considered. In this setting, MET proto-oncogene, receptor tyrosine kinase (MET) exon 14 skipping mutations play a key role in the management of advanced stage NSCLC patients . As a geOverall, MET exon 14 skipping molecular alterations occur in approximately 3–4% of advanced stage NSCLC patients ,10,11,12Due to the heterogeneous nature of the alterations that can lead to the development of MET exon 14 skipping, the different approaches that can be adopted to identify these molecular events remain an open issue. Among these, RNA- or DNA-based next-generation sequencing (NGS), anchored multiplex polymerase chain reaction (PCR), or real-time reverse transcriptase-polymerase chain reaction  methodologies are routinely employed by different laboratories to detect MET exon 14 skipping mutations ,16.Overall, in order to increase the identification of MET exon 14 skipping molecular alterations, and to not leave any patient behind, it is crucial to evaluate inter-laboratory concordance even when different molecular testing platforms are employed. In this setting, the adoption of cell lines with known genomic backgrounds may be a useful tool, as previously demonstrated ,18,19.In this study, we evaluated the concordance among laboratories with high molecular expertise adopting different molecular testing platforms, using artificial reference standards for the detection of the MET exon 14 skipping molecular alteration.n = 10 slides were sent to n = 10 referral laboratories for predictive molecular pathology testing. At each institution, the slides were managed following the center’s internal workflow. After removing the paraffin, the RNA was purified and stored according to the internal testing strategy. The quantity of nucleic acids and, when technically available, the nucleic acids fragmentation index were evaluated. Nucleic acids were analyzed by adopting the routine diagnostic procedures for MET exon 14 skipping of each institution. Within n = 30 working days, the adopted technical assays and all detected molecular alterations (including/Cq/reads count) were listed in a dedicated database and shared with the coordinator center  cell line (Custom METex14 skipping FFPE block) that harbored MET exon 14 skipping developed by Seracare Life Sciences . The artificial control was previously analyzed by the Predictive Molecular Pathology Laboratory at the University of Naples Federico II in order to internally validate the samples before shipping them to other labs. A total of r center .n = 6 slides from the FFPE sample were de-paraffinized and incubated overnight with Proteinase K . Then, the RNA was extracted and purified using the AllPrep DNA/RNA mini kit , according to the manufacturer’s instructions. Moreover, an additional haematoxylin/eosin (H/E) stained slide was digitalized by NanoZoomer 2.0 RS  and archived. RNA quantification and qualification were carried out on the TapeStation 4200 system  using the High Sensitivity RNA Assay on RNA ScreenTape (Agilent Technologies), as previously described [®PGX ready ALK/ROS/RET/MET assay  on the Easy®PGX system, following standardized procedures. The Predictive Molecular Pathology Unit (University of Naples Federico II) received the customized artificial cell block (Custom METex14 skipping FFPE block) to evaluate MET exon 14 skipping mutations with its internal workflow before shipment to other institutions. Briefly, escribed . Data ann = 6/10 (60.0%) and n = 4/10 (40.0%), respectively). For the manual procedures, the RNeasy FFPE kit (Qiagen) assay was adopted by 4 out 6 (66.6%) institutions, while the High Pure miRNA Isolation kit  and the MagMAX™ FFPE DNA/RNA Ultra Kit  were each employed by a single institution, respectively (16.7%). For those utilizing automated procedures, the MagCore Total RNA FFPE One-step kit  with the MagCore Super instrument  and the Maxwell® RSC RNA FFPE Kit  with the Maxwell® RSC instrument  were equally used . Among those using manual procedures, 3 out of 6 (50.0%) centers utilized the Qubit RNA HS Assay Kit (Thermo Fisher Scientific) with the Qubit fluorometer (Thermo Fisher Scientific), 2 out of 6 (33.3%) centers used the NanoDrop™ One/OneC Microvolume UV-Vis Spectrophotometer (Thermo Fisher Scientific), and 1 out 6 (16.7%) centers applied the Ion Library Quantitation kit (Thermo Fisher Scientific) with the QuantStudio 5 Real-Time PCR System (Thermo Fisher Scientific). Moreover, the NanoPhotometer N60 system , NanoDrop™ One/OneC Microvolume UV-Vis Spectrophotometer (Thermo Fisher Scientific), and Quantifluor RNA system  were each adopted by a single institution, respectively, while RNA was not evaluated before molecular analysis in a single case  with an Easy®PGX system; in a single case, MET exon 14 skipping was also confirmed using the Idylla ™ GeneFusion assay  on the Idylla™ platform. Moreover, in 2 out of 5 cases (40.0%), the Oncomine Focus Assay (Thermo Fisher Scientific) with the Io S5™ (center X) and Ion S5™ XL (center X) platforms and the Myriapod® NGS Cancer Panel RNA assay (Diatech Pharmacogenetics S.R.L.) with the iSeq™ and MiSeq™ platforms  were used, respectively. In addition, the Oncomine™ Precision Assay GX  with the fully automated Ion Torrent Genexus System  was used by a single institution. Ion Reporter 5.18.4.0 software was used for variant inspection on samples processed with the Oncomine Focus Assay. In the remaining cases, proprietary analysis software was adopted for data analysis. For the MET exon 14 testing strategy, RNA-based approaches were used in all instances. The RT-PCR-based approach and NGS platforms were equally adopted by receiving institutions . In detail, all RT-qPCR-based institutions employed the EasyAfter the deparaffinization protocol, RNA was directly extracted from the selected slides. In addition, to check the quantity of RNA, two sets of slides representative of the first and last cell-block sections were analyzed in order to verify the neoplastic cell content during FFPE processing. Briefly, RNA was quantified and qualified on both slide sections. Quite similar RNA amounts of 44.6 and 31.0 ng/µL were obtained from the first and second slide set, respectively. In addition, matched RIN values of 2.8 and 2.6 were obtained . ®PGX ready ALK/ROS/RET/MET assay . Data analysis was carried out on the proprietary software of the Easy®PGX platform. A positive result for MET exon 14 skipping in each slide set was reported, as shown in Molecular analysis was successfully performed according to the manual instructions of the EasyAll participating institutions were able to submit their findings prior to the cutoff date. Overall, a median RNA concentration of 24.4 ng/µL (ranging from 4.3 to 51.3 ng/µL) was detected. Particularly, 32.8 ng/µL (ranging from 18.0 to 51.3 ng/µL) and 7.7 ng/µL (ranging from 4.3 to 10.0 ng/µL) RNA were identified in manually and automated nucleic acid extraction workflows, respectively.In all instances, MET exon 14 skipping was successfully identified. Accordingly, molecular analysis showed a median Cq cut off of 30.2 (ranging from 27.1 to 31.4) and 2514 (ranging from 160 to 7526) read counts for RT-qPCR- and NGS-based procedures, respectively. Moreover, the Cq and read count were not reported in a single case, respectively .n = 10 dedicated slides from an artificial FFPE reference standard that harbored the MET exon 14 skipping molecular alteration to n = 10 referral centers involved in molecular testing. Manual and automated nucleic acid extraction protocols were adopted in a comparable number of centers (6 vs. 4). Interestingly, molecular analysis was successfully carried out in all instances without any remarkable differences related to RNA extraction and purification approach. Regarding the molecular analysis of MET exon 14 skipping, 5 out of 10 (50.0%) centers equally adopted RT-PCR and NGS, respectively. Interestingly, no technically relevant issues were observed among the institutions in the detection of MET exon 14 skipping. This point underlines that both applied technical strategies were able to detect MET exon 14 skipping by trained personnel. The predictive role of MET exon 14 skipping for treatment with target drugs has been widely demonstrated. In fact, in prospective clinical trials, tepotinib, capmatinib, and savolitinib showed high activity in advanced stage lung cancer patients harboring the MET exon 14 skipping mutation, including treatment-naïve patients and those affected by pulmonary sarcomatoid carcinoma [This study aimed to evaluate the technical feasibility of analyzing MET exon 14 skipping on customized artificial samples using different genomic platforms. Particularly, this multicenter observational study tried to highlight the robustness of technical workflows adopted by each participating center in the detection of the aberrant MET transcript for targeted therapy of selected NSCLC patients ,9. Preanarcinoma ,20. Intearcinoma ,22,23,24arcinoma . Overallarcinoma ,25,26. Harcinoma . This laarcinoma . For exaarcinoma . Overallarcinoma . The hybarcinoma . This pharcinoma . In addiarcinoma . Howeverarcinoma . It was arcinoma . In partarcinoma .The use of artificial controls was a useful tool to evaluate inter- and intra-laboratory reproducibility, as was previously demonstrated in experiments carried out by the Molecular Cytopathology Meeting Group. In this study, the adoption of an engineered cell line harboring a known genomic alteration that could be distributed to different laboratories was fundamental in the validation of molecular platforms not only for DNA-based but also for RNA-based biomarkers ,18,19. F